Cross linked hyaluronic acid-silk and uses thereof

ABSTRACT

The present specification provides for methods for purifying fibroins, purified fibroins, methods of conjugating biological and synthetic molecules to fibroins, fibroins conjugated to such molecules, methods of making fibroin hydrogels, fibroin hydrogels and fibroin hydrogel formulations useful for a variety of medical uses, including, without limitation uses as bulking agents, tissue space fillers, templates for tissue reconstruction or regeneration, cell culture scaffolds for tissue engineering and for disease models, surface coating to improve medical device function, or drug delivery devices.

CROSS REFERENCE

This patent application is a continuation of U.S. patent applicationSer. No. 12/764,052, filed Apr. 20, 2010, which claims priority to U.S.Provisional Patent Application Ser. No. 61/170,895 filed Apr. 20, 2009,each of which are hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present specification discloses purified silk fibroin and method forpurifying silk fibroins, hydrogels comprising silk fibroin with orwithout an amphiphilic peptide and methods for making hydrogelscomprising silk fibroin and the use of silk fibroin hydrogels in avariety of medical uses, including, without limitation fillers fortissue space, templates for tissue reconstruction or regeneration,scaffolds for cells in tissue engineering applications and for diseasemodels, a surface coating to improve medical device function, or as aplatform for drug delivery.

BACKGROUND

Silk refers to a filamentous product secreted by an organism such as aspider or silkworm. Fibroin is the primary structural component of silk.It is composed of monomeric units comprising an about 350 kDa heavychain and an about 25 kDa light chain, and interspersed within thefibroin monomers is another about 25 kDa protein derived from the P25gene. The ratio of heavy chain:light chain:P25 protein is about 6:6:1.Fibroin is secreted by the silk glands of the organism as a pair ofcomplementary fibrils called “brins”. As fibroin brins leave the glands,they are coated with sericin, a glue-like substance which binds thebrins together. Sericin is often antigenic and may be associated with anadverse tissue reaction when sericin-containing silk is implanted invivo.

Silkworm silk fibers traditionally available in the commercial marketare often termed “degummed”, which refers to the loosening and removalof a portion of the sericin coat surrounding the two fibroin brinsthrough washing or extraction in hot soapy water. This degummed silkoften contains or is recoated with sericin and other impurities in orderto bind the plied multifilament together into a single fiber. Therefore,degummed silk, unless explicitly stated to the contrary, typicallycontains twenty percent to twenty-eight percent (by weight) sericin andcan not be assumed to be sericin-free.

Silk fibers have historically been valued in surgery for theirmechanical properties, particularly in the form of braided filamentsused as a suture material. Residual sericin that may be contained inthese materials stands as a potential obstacle to its use as abiomaterial as it does present the possibility for a heightened immuneresponse. This sericin contamination may be substantially removedthough, resulting in a virtually sericin-free fibroin which may be usedeither as fibers or dissolved and reconstituted in a number of forms.For example, natural silk from the silkworm Bombyx mori may be subjectedto sericin extraction, spun into yarns then used to create a matrix withhigh tensile strength suitable for applications such as bioengineeredligaments and tendons. Use of regenerated silk materials has also beenproposed for a number of medical purposes including wound protection,cell culture substrate, enzyme immobilization, soft contact lenses, anddrug-release agents.

Silk fibroin devices whether native, dissolved, or reconstituted, do nottypically contain cell-binding domains such as those found in collagen,fibronectin, and many other extracellular matrix (ECM) molecules.Fibroin is also strongly hydrophobic due to the β-sheet-rich crystallinenetwork of the core fibroin protein. These two factors couple toseverely limit the capacity of native host cells to bind to and interactwith implanted silk devices, as neither inflammatory cells likemacrophages or reparative cells like fibroblasts are able to attachstrongly, infiltrate and bioresorb the silk fibroin devices. In the caseof virgin silk and black braided (wax or silicone coated) silk sutures,this is typically manifested in a harsh foreign-body response featuringperipheral encapsulation. Substantially sericin-free silk experiences asimilar, though substantially less vigorous response when implanted. Inessence, the host cells identify silk as a foreign body and opt to wallit off rather than interact with it. This severely limits the subsequentlong-term potential of the device particularly relating to tissuein-growth and remodeling and potentially, the overall utility of thedevice. If it is possible to provide a more effective biomaterialformulation for mediating host-device interactions whereby cells areprovided with a recognizable, acceptable and hence biocompatiblesurface, the biological, medicinal and surgical utility of silk isdramatically improved.

One possible means of introducing this improved cell-materialinteraction is to alter the silk fibroin material format into a morebiocompatible matrix. Manipulating the silk fibroin to make it into asilk hydrogel formulation is one particularly intriguing option becauseit consists of a silk protein network which is fully saturated withwater, coupling the molecular resiliency of silk with thebiocompatibility of a “wet” material. Generation of a silk hydrogel maybe accomplished in short by breaking apart native silk fibroin polymersinto its individual monomeric components using a solvent species,replacing the solvent with water, then inducing a combination of inter-and intra-molecular aggregation. It has been shown that the sol-geltransition can be selectively initiated by changing the concentration ofthe protein, temperature, pH and additive (e.g., ions and hygroscopicpolymers such as poly(ethylene oxide) (PEO), poloxamer, and glycerol).Increasing the silk concentration and temperature may alter the timetaken for silk gelation by increasing the frequency of molecularinteractions, increasing the chances of polymer nucleation. Anothermeans of accelerating silk gelation is through use of calcium ions whichmay interact with the hydrophilic blocks at the ends of silk moleculesin solution prior to gelation. Decreasing pH and the addition of ahydrophilic polymer have been shown to enhance gelation, possibly bydecreasing repulsion between individual silk molecules in solution andsubsequently competing with silk fibroin molecules in solution for boundwater, causing fibroin precipitation and aggregation.

Other silk fibroin gels have been produced by, for example, mixing anaqueous silk fibroin solution with protein derived biomaterials such asgelatin or chitosan. Recombinant proteins materials based on silkfibroin's structure have also been used to create self-assemblinghydrogel structures. Another silk gel, a silk fibroin-poly-(vinylalcohol) gel was created by freeze- or air-drying an aqueous solution,then reconstituting in water and allowing to self-assemble. Silkhydrogels have also been generated by either exposing the silk solutionto temperature condition of 4° C. (Thermogel) or by adding thirtypercent (v/v) glycerol (Glygel). Silk hydrogels created via afreeze-thaw process have not only been generated but also used in vitroas a cell culture scaffold.

The use of silk hydrogels as biomaterial matrices has also been exploredin a number of ways. General research on hydrogels as platforms for drugdelivery, specifically the release behavior of benfotiamine (a syntheticvariant of vitamin B₁) coupled to silk hydrogel was investigated. Thestudy revealed both silk concentration and addition of other compoundsmay factor in to the eventual release profile of the material.Similarly, the release of FITC-labeled dextran from a silk hydrogelcould be manipulated by altering the silk concentrations within the gel.

Further studies of silk hydrogels have been performed in vivo as well.For example, the material has been used in vivo to provide scaffoldingfor repair of broken bones in rabbits and showed an accelerated healingrate relative to control animals. Of particular interest, the in situstudy also illustrated that the particular formulation of silk hydrogeldid not elicit an extensive immune response from the host.

Despite early promise with silk hydrogel formulations in vivo, sericincontamination remains a concern in their generation and use just as withnative fibroin for reasons of biocompatibility as well as the potentialfor sericin to alter gelation kinetics. The existence of sericinmolecules in the silk solution intermediate prior to gelation may alsocompromise final gel structural quality, i.e., the distribution ofβ-sheet structure. For these reasons the removal of sericin from silkfibroin material prior to hydrogel manufacture remains a concern. Thepotential for disruption of gelation kinetics and structure bycontaminants also presents the need for development of a process whichconsistently ensures structural uniformity and biocompatibility.

SUMMARY OF THE INVENTION

The embodiments described herein provide for silk hydrogel formulationsthat may be useful for a variety of medical uses. More specifically,example embodiments of the present invention provide for gels includingsilk fibroin and peptides. Other example embodiments provide for the useof organic enhancers which improve device utility and functional peptideenhancers that may improve utility and biocompatibility of silkformulations. Silk hydrogel embodiments may be used as tissue spacefillers, templates for tissue reconstruction or regeneration, cellculture scaffolds for tissue engineering and for disease models, surfacecoating to improve medical device function, or drug delivery devices.

One embodiment provides for an injectable silk gel comprising a gelphase and a carrier phase (which may provide additional lubricity) inwhich the gel phase comprises water, substantially sericin-depleted silkfibroin and an amphiphilic peptide. In another embodiment, the gel phaseis about 1% to 99%, for example the gel phase is about 50% to about 99%of the total formulation volume with the carrier phase providing theremainder. For example, the gel phase is about 75% of the totalformulation volume and the carrier phase is the remaining 25%. The gelphase may comprise about 0.5% to about 20% silk fibroin protein by mass,for example about 1% to about 10%, or about 4% to about 6%. In oneembodiment, the silk fibroin comprises about 0.5% to about 9.9% of thetotal formulation mass.

In a particular embodiment, the peptide is an amphiphilic peptideconsisting of a tail region, followed by a spacer region and finally thesequence arginine-glycine-aspartic acid, known as the RGD motif. Forexample, the total peptide is 23 amino acids in length (hereinafter,referred to as “23RGD”). The gel phase may comprise, for example, amolar ratio of about 1:100 moles to about 100:1 moles of this peptideper mole of silk fibroin.

Another example embodiment provides for an injectable gel formulationcomprising silk fibroin and an amphiphilic peptide, wherein theformulation comprises from about 1% about 20%, for example about 4% toabout 6% silk fibroin, and the amphiphilic peptide is 23RGD.

Yet another embodiment provides for an injectable gel formulationcomprising silk fibroin and 23RGD, wherein the formulation comprisesfrom about 4% to about 6% silk fibroin, and 23RGD concentration is 3:1moles 23RGD/mole silk.

Another particular embodiment provides for an implantable gelformulation comprising silk fibroin and the 23RGD wherein the gelformulation comprises from about 4% to about 8% silk fibroin and the23RGD concentration is about 1:10 to 10:1 moles of 23RGD per mole ofsilk fibroin.

In another embodiment, the gel phase comprises a protein structureconsisting predominantly of the β-sheet conformation with components ofα-helix, random coil, and unordered structures.

Another example embodiment of invention relates to a kit including asterile silk gel formulation packaged in a 1 mL syringe with a 26 gneedle and blended with a material commonly referred to as a “localanesthetic”. This anesthetic might be more specifically lidocaine.Dependent upon application, the kit includes syringes sizes from 0.5 mLto 60 mL, where applications requiring larger volumes (e.g., bonefillers, disc fillers) are supplied in a larger size syringe.Additionally, needle gage is adjusted according to injection site withan acceptable range of 10 g to 30 g needles. For example, 26 g to 30 gneedles are used for intradermal injections. Furthermore, the localanesthetic is not blended into the formulation for applications wherethe anesthetic is preferably applied separately or applications forwhich an anesthetic is not needed.

In another embodiment, the silk gel formulation is processed in a batchsystem by obtaining an 8% silk solution, adding ethanol/23RGD togenerate a firm 4%-6% gel, allowing this to stand for at least 24 hours.The gel is then rinsed in water to remove residual free gelation agents(both 23RGD and ethanol), adding saline solution to the gel as a carrierphase and developing a homogeneous suspension. Suspensionviscosity/injectability is then tailored by manipulating gelconcentration, particle size, and saline content, milling the gel to adesired particle size that makes the gel injectable through a needle(for example a 30 g needle), loading the gel into a syringe, andsterilizing the gel with gamma irradiation.

In another aspect, the injectable formulation includes a gel comprisingsubstantially sericin-depleted silk fibroin and an amphiphilic peptideand a carrier phase, wherein the formulation, upon injection, remainssubstantially at the injection site for about two weeks to about sixtymonths depending upon a desired application. For example, oneformulation, for soft tissue filling may employ a 1%-6% silk gel with20%-50% saline carrier at an average particle size of 20 μm-30 μm, andbe deliverable through a 26 g-30 g needle with ˜5N of force whileremaining substantially for one month to nine months at the injectionsite. One example formulation for hard tissue filling may employ a6%-10% silk gel with 0%-25% saline carrier at a 50 μm-1000 μm particlesize, and be deliverable through a 10 g-18 g needle at ˜5N of forcewhile remaining substantially for nine to fifteen months at theinjection site.

In one embodiment, the present invention provides a five-amino acidpeptide “tail” capable of linking or conjugating a molecule X to a silkmolecule or fibroin when the molecule X is attached to the tail. In oneembodiment, the tail peptide comprises of hydrophobic and/or apolaramino acid residues. In another embodiment, the tail peptide comprisesof amino acid residues capable of hydrogen bonding and/or covalentbonding. In other embodiments, the tail peptide comprises any of thetwenty conventional standard amino acid residues.

In one embodiment, the five-amino acid peptide “tail” comprises aminoacid residues that are part hydrophobic (i.e. the part of the side-chainnearest to the protein main-chain), for e.g. arginine and lysine.

In one embodiment, the five-amino acid peptide “tail” is separated froma molecule X by a spacer peptide. The length of the space peptide can beof variable length.

In one embodiment, the molecule X is any biological molecule or fragmentthereof. In other embodiments, the molecule X is any recombinant,synthetic, or non-native polymeric compounds. Basically, a molecule X isany entity, natural or synthetic, that can be useful and can be use inthe context of silk hydrogels.

In one embodiment, the present invention provides a synthetic moleculehaving the formula: (molecule X)n-(spacer peptide)₀₋₃₀₀-(tail)-NH₂ forlinking with silk molecule or fibroin, wherein “n” is a whole integerranging from 1-30, and wherein the amino acid residues of the spacerranges from 0-300.

In one embodiment, the invention provides a method of conjugating amolecule X to a silk molecule or fibroin comprising mixing a syntheticmolecule having the formula: (molecule X)n-(spacerpeptide)₀₋₃₀₀-(tail)-NH₂ with a silk molecule or fibroin or silksolution, wherein “n” is a whole integer ranging from 1-30, and whereinthe amino acid residues of the spacer ranges from 0-300.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B illustrate the impact of 23RGD on the gelation times of silkhydrogels manufactured under various circumstances for example withoutenhancers or with a water/23RGD enhancer (FIG. 1A), or with an ethanolenhancer or combined ethanol-23RGD enhancers (FIG. 1B). Depending uponthe ratio of 23RGD to silk used and the specific enhancer solvents, thepeptide may function as either an accelerant or decelerant of theprocess.

FIG. 2 is a graph of HPLC data illustrating the integration of 23RGD andstability of its binding to 4% silk gel material made with an enhancersolution consisting of a 3:1 molar ratio of 23RGD:silk dissolved in 90%ethanol, 10% water when rinsed multiple times in ultra-purified waterover several days. Data are shown for both total peak area andcalculated 23RGD:silk molar ratio based on a 23RGD standard curve.

FIG. 3 is a graph comparing gel dry mass component at different RGDconcentrations for 2% silk gels (A), 4% silk gels (B), and 6% gels(C). * Samples differ significantly, p<0.05; † sample differssignificantly from all others; ‡ all samples differ significantly.

FIG. 4 illustrates the impact upon silk hydrogel water absorption andretention as identified in a gel drying assay. Data are shown as thepercentage of mass retained by a silk gel sample (n=4 for each type)after being subjected to a 96-hour lyophilization process. Increasingconcentrations of 23RGD enhancer caused increasing dry mass in the gelmaterials more substantial than the mass of the peptide itself. Thisphenomenon is likely due to structural differences in 23RGD-enhancedgels which do not permit a level of water entrainment equal to those ofgels enhanced only with ethanol.

FIG. 5 shows a comparison of the percent mass loss over time due tobioresorption of samples cast by PG and EEG methods (A), cast fromincreasing silk concentrations (B), and cast using increasing RGDconcentrations (C). * Samples differ significantly, p<0.05; † samplediffers significantly from all others; ‡ all samples differsignificantly.

FIG. 6 illustrates wet mass loss due to proteolytic bioresorption ofsilk hydrogels enhanced by a combination of 23RGD and ethanol atincreasing concentrations of 23RGD. As a general trend, gels enhancedwith 23RGD tend to be bioresorbed more quickly based upon this assay.

FIG. 7 is a second illustration of the bioresorption behavior of23RGD-enhanced and non-23RGD-enhanced silk hydrogels when incubated in aprotease solution. This bioresorption data serves to reinforce thetrend, illustrated in FIG. 5, of a slightly more rapid rate ofbioresorption of 23RGD-enhanced hydrogels in comparison tonon-23RGD-enhanced gels. The figure also supports the more thoroughremoval of α-helix and random coil conformations from 23RGD-enhancedgels in FIG. 6 over four days of incubation in protease.

FIGS. 8A-8E show structural features observed by Fourier-TransformInfrared (FTIR) spectroscopy of 4% silk fibroin hydrogel devices whichare enhanced by ethanol alone, and two 23RGD-ethanol enhancers. The fullspectra (FIG. 9A) of the materials are compared and the Amide I Band(1700-1600 cm⁻¹) highlighted for particular attention (FIG. 9B) becauseof its relevance to secondary protein structure. Of specific interest isthe commonality between all gels in their rich β-sheet structure (1700cm⁻¹ and 1622 cm⁻¹ respectively, highlighted in FIG. 9C) at all-timepoints. These peaks become more pronounced after bioresorption, andbegin to differentiate 23RGD-enhanced materials from materials enhancedwith ethanol alone. This is evidenced in 23RGD-enhanced gels by a peakshift to lower wave numbers by the 1622 cm⁻¹ peak and dramaticallyincreased prominence of the 1700 cm⁻¹ peak. Additional differencesbetween bioresorbed and non-bioresorbed gels may be seen in regions ofthe spectrum known to correlate to α-helix and random coil conformations(1654 cm⁻¹ and 1645 cm⁻¹ respectively highlighted in FIG. 9C). Theseconformations are extensively digested in all gel types, but mostcompletely in gels enhanced by 23RGD. This suggests that 23RGD-enhancedgels tend to bioresorb to a very β-sheet rich secondary structure in amore rapid fashion than non-23RGD-enhanced gels. Spectra shown werecollected on a Bruker Equinox 55 FTIR unit using a compilation of 128scans with a resolution of 4 cm⁻¹.

FIG. 9 shows a comparative FTIR spectra illustrating the effects ofdiffering gelation techniques on gel protein structure before (Day 0)and after (Day 4) proteolytic bioresorption. Groups assessed includedsamples cast by PG and EEG methods (A), cast from increasing silkconcentrations (B), and cast using increasing RGD concentrations (C).

FIG. 10 shows representative micrographs of H&E-stained histologicalsections collected from silk gels implanted subcutaneously in rats.Samples of 4% silk fibroin hydrogel formed by passive gelation (4P), 4%silk fibroin hydrogel formed by ethanol-enhanced gelation (4E), and 6%silk fibroin hydrogel formed by ethanol-enhanced gelation (6E) werecompared at 7 days (A, B, and E respectively) with 4E and 6E samplescompared again at days 28 (C and F) and 57 (D and G).

FIG. 11 shows representative gross photographs of 8% silk fibroinhydrogel devices both unmodified (A) and 23RGD-enhanced (D) after atwo-week subcutaneous incubation in Lewis rats. Also shown aremicrographs resultant from H & E stains of the unmodified (B and C) and23RGD-coupled (E and F) samples at 10× and 20× magnification. Thesegross images coupled with the histological micrographs provide evidenceof a less extensive inflammatory response during early deviceintegration being associated with 23RGD-enhanced gel thannon-23RGD-enhanced gel.

FIG. 12 shows representative histology collected from a thirteen-weekstudy of 4% 3:1 23RGD-enhanced silk hydrogel blended with 25% saline(left panels, H&E stain Trichrome stain) and ZYPLAST™ (right panels H&Estain, Trichrome stain) and injected into the intradermis of guinea pig.Each material type exhibited some clear evidence of implanted device in75% of their respective implant sites. These micrographs indicate strongsimilarities not only between the long-term bioresorptioncharacteristics but also long-term host tissue response betweencollagen-derived biomaterials and this particular 23RGD-enhanced silkhydrogel formulation.

FIG. 13 shows representative micrographs of H&E-stained histologicalsections collected from Day 28 explants of 4% silk fibroin, 10% saline(A); 4% silk fibroin, 1:1 23RGD, 10% saline (B); 6% silk fibroin, 1:123RGD, 10% saline (C); ZYPLAST™ (D); 4% silk fibroin, 25% saline (E); 4%silk fibroin, 1:1 23RGD, 25% saline (F); 6% silk fibroin, 10% saline(G); HYLAFORM™ (H); 6% silk fibroin, 25% saline (I); 4% silk fibroin,3:1 23RGD, 25% saline (J); and 6% silk fibroin, 1:1 23RGD, 25% saline(K).

FIG. 14 shows representative micrographs of Day 92 histological sectionsof 4% silk fibroin, 3:1 23RGD, 25% saline (A-D) and ZYPLAST™ samples(E-H) stained with H&E at 4× (A and E), 10× (B and F), stained withMasson's Trichrome at 10× (C and G) and under polarized light at 10× (Dand H).

FIG. 15 is a photograph of a custom-built testing jig used inconjunction with an Instron 8511 (Instron Corporation, Canton Mass.) inconjunction with Series IX software and a 100 N load cell forcharacterizing the injection forces associated with forcing silk gelthrough a 30 g needle.

FIG. 16 illustrates the average extrusion force data from mechanicaltesting of various silk gel formulations illustrating the effects ofchanging comminution method (A), saline concentration (B), silkconcentration (C), and RGD content (D). Values are reported as anaverage of n=4 tests at each displacement rate with standard deviationillustrated as error bars. * Samples differ significantly, p<0.05; †sample differs significantly from all others in group at same strainrate; ‡ all samples in group differ significantly from all others ingroup at same strain rate.

FIG. 17 shows representative ESEM micrographs of selectedRGD/ethanol-induced silk precipitates generated from the previouslymentioned formulations. BASE (A), SCVLO (B), RHI (C), RLO (D), AVHI (E),ECLO (F), AVLO (G), and 3R 6.7:1 (H) are shown at 200× magnification.

FIGS. 18A-18D show a comparison of the total dry mass of precipitaterecovered from each silk precipitate formulation (n=4 for each type)after being subjected to a 96-hour lyophilization process. Data aregrouped to compare the effects of changing volume ratio of accelerantadded (A), concentration of 23RGD in the accelerant (B), changing theinitial silk concentration (C), and changing the concentration ofethanol in the accelerant (D). It was shown that increasing any of thesevolumes or concentrations resulted in greater quantities of precipitate,though none appear to have substantially greater impact than another.This phenomenon is likely due to basic kinetics of the assemblyreaction, with each reagent in turn appearing both as an excess and aslimiting dependent upon the specific formulation. *—significantdifference, p<0.05; †—Group differs significantly from all others.

FIGS. 19A-19D show a comparison of the percentage of dry mass in each ofprecipitate recovered (n=4 for each type) after being subjected to a96-hour lyophilization process. Data are grouped to compare the effectsof changing volume ratio of accelerant added (A), concentration of 23RGDin the accelerant (B), changing the initial silk concentration (C), andchanging the concentration of ethanol in the accelerant (D). Increasingthe concentration of 23RGD used increased the dry mass percentage ofprecipitates, while increasing the ethanol percentage in the accelerantdecreased dry mass. These changes may stem from formation of altered gelnetwork structures caused by manipulation of these variables, likelymore crystalline in the case of 23RGD increases and less crystalline inthe case of ethanol concentration increases. *—significant difference,p<0.05; †—Group differs significantly from all others.

FIG. 20 shows representative FTIR spectra of the Amide I band for23RGD/ethanol-induced silk precipitates immediately after processing(D0). Spectra are grouped to compare the effects of changing volumeratio of accelerant added (A), concentration of 23RGD in the accelerant(B), changing the initial silk concentration (C), and changing theconcentration of ethanol in the accelerant (D). These spectra illustratethat similarities exist between all groups although changing 23RGDconcentrations and ethanol concentrations may substantially impactprecipitate structure. Increasing concentrations of decreased β-sheetseen in a peak shift from ˜1621 cm⁻¹ in RVLO to ˜1624 cm⁻¹ in RLO. Afurther increase in 23RGD concentration in both BASE and RHI caused thisweakened β-sheet again along with increased signal values in the 1654cm¹ and 1645 cm¹ range, correlating to increased random coil andα-helical content. An increased percentage of ethanol decreased thecontent of α-helical and random coil shown by decreased signal between1670 cm⁻¹ and 1630 cm⁻¹ in both ECLO and BASE samples relative to ECVLO.This decrease in α-helical and random coil is accompanied by an increasein β-sheet structure. The findings relating to 23RGD and ethanolconcentrations reinforce the trends observed in the percent dry mass ofthe precipitates, supposing that α-helical and random coil motifsentrain more water than β-sheet regions.

FIG. 21 is a representative micrograph of Congo red-stained23RGD/ethanol-induced silk precipitates under polarized light at 20×magnification. A lack of emerald-green birefringence indicates anegative result in testing for amyloid fibril formation.

FIG. 22 shows comparison of 23RGD:silk molar ratio in each ofprecipitate recovered. Data are grouped to compare the effects ofchanging volume ratio of accelerant added (A), concentration of 23RGD inthe accelerant (B), changing the initial silk concentration (C), andchanging the concentration of ethanol in the accelerant (D). Inexamining the 23RGD bound to the precipitates, all materials containedmore 23RGD than predicted by initial calculations aside of AVHI, RVLO,RHI, and SCVLO. In the cases of AVHI and ECLO the 23RGD quantity wassubstantially more than was expected. In the cases of BASE, RLO, SCVLO,and SCLO the 23RGD quantities were approximately double that expected.This may be indicative of the formation of a 23RGD dimer in the 90%ethanol accelerant solution. The RVLO samples were made with a 23RGDconcentration of 0.49 mg/mL in the accelerant, the lowest used in thisstudy and potentially within the solubility range of 23RGD in 90%ethanol. RLO samples used 1.47 mg/mL and most other formulations weremade with a 23RGD accelerant concentration of 2.45 mg/mL, above the23RGD concentration at which dimerization became favorable in thesolution. Further highlighting the possibility of 23RGD dimerizing inthe ethanol solution is the behavior of ECLO precipitation. The 23RGDconcentration remains 2.45 mg/mL as with BASE and AVLO but the waterconcentration in the accelerant is increased to 20% and results in abinding of about 1.5-fold the expected total of 23RGD instead of 2-fold.This may be due to dis-solution of a greater quantity of 23RGD, causingcoexistence between dimeric and monomeric 23RGD in solution reflected inthe subsequent binding ratios. *—significant difference, p<0.05; †—Groupdiffers significantly from all others; ‡—All groups differsignificantly.

FIGS. 23A-23D show a representative FTIR spectra of the Amide I band areshown for 23RGD/ethanol-induced silk precipitates initially (D0) andafter proteolytic bioresorption (D2). Spectra are grouped to compare theeffects of changing volume ratio of accelerant added (A), concentrationof 23RGD in the accelerant (B), changing the initial silk concentration(C), and changing the concentration of ethanol in the accelerant (D).Accelerant quantity added did not substantially affect the bioresorptionbehavior of the materials as BASE, AVHI and AVLO all featured decreasedlevels of α-helix and random coil motifs. This decrease was slightlylarger in the case of AVLO which also featured a peak shift from 1624cm⁻¹ to 1622 cm⁻¹, indicating a more stable β-sheet structure. 23RGDconcentration did not appear to affect bioresorption behavior of thematerials either as RVLO, RLO, BASE and RHI all showed decreased inα-helix and random coil motifs, though a greater portion of α-helix andrandom coil remained intact in RHI. Silk concentration did notsubstantially affect the bioresorption behavior of the materials as BASEand SOLO exhibited decreased levels of α-helix and random coil motifsand featured slight peak shifts from 1624 cm⁻¹ to 1623 cm⁻¹.

DETAILED DESCRIPTION OF INVENTION

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

As used herein and in the claims, the singular forms “a,” “an,” and“the” include the plural reference unless the context clearly indicatesotherwise. Thus, for example, the reference to a peptide is a referenceto one or more such peptides, including equivalents thereof known tothose skilled in the art. Other than in the operating examples, or whereotherwise indicated, all numbers expressing quantities of ingredients orreaction conditions used herein should be understood as modified in allinstances by the term “about.”

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood to one of ordinaryskill in the art to which this invention pertains. Although any knownmethods, devices, and materials may be used in the practice or testingof the invention, the methods, devices, and materials in this regard aredescribed here.

As used herein, the term “about” means that the item, parameter or termso qualified encompasses a range of plus or minus ten percent above andbelow the value of the stated item, parameter or term.

Aspects of the present specification provide, in part, a depolymerizedsilk fibroin. As used herein, the term “depolymerized silk fibroin” issynonymous with “dissolved silk” and “dissolved silk fibroin” and refersto silk fibroin existing primarily as monomers or other lower oligomericunits. Treatment of naturally-occurring fibrous silk with a dissolutionagent, such as, e.g., a chaotropic agent results in depolymerized silkfibroin. The depolymerized silk fibroin used for preparing silk fibroinhydrogel is an intermediate in the silk hydrogel production process anda direct precursor to the hydrogel material. The depolymerized silkfibroin can be made from raw cocoons, previously degummed silk or anyother partially cleaned silk. This may also include material commonlytermed as “waste” from the reeling process, i.e. short fragments of rawor degummed silk, the sole precaution being that the silk must besubstantially cleaned of sericin prior to making fibroin solution andinducing gel formation. A particular source of raw silk is from commondomesticated silkworm B. mori, though several other sources of silk maybe appropriate. This includes other strains of Bombycidae includingAntheraea pernyi, Antheraea yamamai, Antheraea mylitta, Antheraeaassama, and Philosamia cynthia ricini, as well as silk producing membersof the families Saturniidae, Thaumetopoeidae, and silk-producing membersof the order Araneae. The material may also be obtained from otherspider, caterpillar, or recombinant sources.

Aspects of the present specification provide, in part, a polymerizedsilk fibroin. As used herein, the term “polymerized silk fibroin” issynonymous with “silk fibroin” and refers to silk fibroin existingprimarily as a polymer. A polymerized silk fibroin or silk fibroin ismade by, e.g., a gelation process disclosed in the presentspecification.

The hydrogels and formulations disclosed in the present specificationprovide for a depolymerized silk fibroin and/or silk fibroin that issubstantially free of sericin. Methods for performing sericin extractionhave been described in pending U.S. patent application Ser. No.10/008,924, Publication No. 20030100108, Matrix for the production oftissue engineered ligaments, tendons and other tissue, published May 29,2003. That application refers to cleaned fibroin fibers spun into yarns,used to create a porous, elastic matrix suitable as a substrate forapplications requiring very high tensile strength, such as bioengineeredligaments and tendons.

Extractants such as urea solution, hot water, enzyme solutions includingpapain among others which are known in the art to remove sericin fromfibroin would also be acceptable for generation of the silk. Mechanicalmethods may also be used for the removal of sericin from silk fibroin.This includes but is not limited to ultrasound, abrasive scrubbing andfluid flow. The rinse post-extraction is conducted preferably withvigorous agitation to remove substantially any ionic contaminants,soluble, and in soluble debris present on the silk as monitored throughmicroscopy and solution electrochemical measurements. A criterion isthat the extractant predictably and repeatably remove the sericin coatof the source silk without significantly compromising the molecularstructure of the fibroin. For example, an extraction may be evaluatedfor sericin removal via mass loss, amino acid content analysis, andscanning electron microscopy. Fibroin degradation may in turn bemonitored by FTIR analysis, standard protein gel electrophoresis andscanning electron microscopy.

In certain cases, the silk utilized for generation of a silk hydrogelhas been substantially depleted of its native sericin content (i.e., ≦4%(w/w) residual sericin in the final extracted silk). Alternatively,higher concentrations of residual sericin may be left on the silkfollowing extraction or the extraction step may be omitted. In aspectsof this embodiment, the sericin-depleted silk fibroin has, e.g., about1% (w/w) residual sericin, about 2% (w/w) residual sericin, about 3%(w/w) residual sericin, or about 4% (w/w) residual sericin. In otheraspects of this embodiment, the sericin-depleted silk fibroin has, e.g.,at most 1% (w/w) residual sericin, at most 2% (w/w) residual sericin, atmost 3% (w/w) residual sericin, or at most 4% (w/w) residual sericin. Inyet other aspects of this embodiment, the sericin-depleted silk fibroinhas, e.g., about 1% (w/w) to about 2% (w/w) residual sericin, about 1%(w/w) to about 3% (w/w) residual sericin, or about 1% (w/w) to about 4%(w/w) residual sericin.

In certain cases, the silk utilized for generation of a silk hydrogel isentirely free of its native sericin content. As used herein, the term“entirely free (i.e. “consisting of” terminology) means that within thedetection range of the instrument or process being used, the substancecannot be detected or its presence cannot be confirmed.

In certain cases, the silk utilized for generation of a silk hydrogel isessentially free of its native sericin content. As used herein, the term“essentially free” (or “consisting essentially of”′) means that onlytrace amounts of the substance can be detected.

Additionally, the possibility exists for deliberately modifying hydrogelproperties through controlled partial removal of silk sericin ordeliberate enrichment of source silk with sericin. This may function toimprove hydrogel hydrophilicity and eventual host acceptance inparticular biological settings despite sericin antigenicity.

After initial degumming or sericin removal from fibrous silk used forgeneration of a hydrogel, the silk is rinsed free of gross particulatedebris. It is of concern to remove such particles as either solvent(i.e., specific solvent of interest for device generation) soluble orinsoluble compounds may profoundly affect the outcome of the hydrogelgenerated from the intermediate solution. Insoluble compounds may serveas nucleation points, accelerating the gelation phenomenon andpotentially altering subsequent hydrogel protein structure. Solublecompounds may also serve to interface with the protein network of thehydrogel, altering the organizational state of the device. Either typeof compound could also compromise biocompatibility of the device.

Prior to dissolution, the prepared silk may be subjected to associationof various molecules. The binding between these compounds and the silkmolecules may be unaffected by the dissolving agent used for preparationof silk solution intermediate. The method for coupling the modifyingcompound to the prepared silk may vary dependent upon the specificnature of the bond desired between silk sequence and the modifier.Methods are not limited to but may include hydrogen bonding throughaffinity adsorption, covalent crosslinking of compounds or sequentialbinding of inactive and active compounds. These molecules may include,but would not be limited to, inorganic compounds, peptides, proteins,glycoproteins, proteoglycans, ionic compounds, natural, and syntheticpolymers. Such peptides, proteins, glycoproteins and proteoglycans mayinclude classes of molecules generally referred to as “growth factors”,“cytokines”, “chemokines”, and “extracellular matrix compounds”. Thesecompounds might include such things as surface receptor binding motifslike arginine-glycine-aspartic acid (RGD), growth factors like basicfibroblast growth factor (bFGF), platelet derived growth factor (PDGF),transforming growth factor (TGF), cytokines like tumor necrosis factor(TNF), interferon (IFN), interleukins (IL), and structural sequencesincluding collagen, elastin, hyaluronic acid and others. Additionallyrecombinant, synthetic, or non-native polymeric compounds might be usedas decoration including chitin, poly-lactic acid (PLA), andpoly-glycolic acid (PGA). Other compounds linked to the material mayinclude classes of molecules generally referred to as tracers,contrasting agents, aptamers, avimers, peptide nucleic acids andmodified polysaccharide coatings.

For example, the initially dissolved silk may be generated by a 4 hourdigestion at 60° C. of pure silk fibroin at a concentration of 200 g/Lin a 9.3 M aqueous solution of lithium bromide to a silk concentrationof 20% (w/v). This process may be conducted by other means provided thatthey deliver a similar degree of dissociation to that provided by a 4hour digestion at 60° C. of pure silk fibroin at a concentration of 200g/L in a 9.3 M aqueous solution of lithium bromide. The primary goal ofthis is to create uniformly and repeatably dissociated silk fibroinmolecules to ensure similar fibroin solution properties and,subsequently, device properties. Less substantially dissociated silksolution may have altered gelation kinetics resulting in differing finalgel properties. The degree of dissociation may be indicated byFourier-transform Infrared Spectroscopy (FTIR) or x-ray diffraction(XRD) and other modalities that quantitatively and qualitatively measureprotein structure. Additionally, one may confirm that heavy and lightchain domains of the silk fibroin dimer have remained intact followingsilk processing and dissolution. This may be achieved by methods such asstandard protein sodium-dodecyl-sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) which assess molecular weight of theindependent silk fibroin domains.

System parameters which may be modified in the initial dissolution ofsilk include but are not limited to solvent type, silk concentration,temperature, pressure, and addition of mechanical disruptive forces.Solvent types other than aqueous lithium bromide may include but are notlimited to aqueous solutions, alcohol solutions,1,1,1,3,3,3-hexafluoro-2-propanol, and hexafluoroacetone,1-butyl-3-methylimidazolium. These solvents may be further enhanced byaddition of urea or ionic species including lithium bromide, calciumchloride, lithium thiocyanate, zinc chloride, magnesium salts, sodiumthiocyanate, and other lithium and calcium halides would be useful forsuch an application. These solvents may also be modified throughadjustment of pH either by addition of acidic of basic compounds.

Further tailoring of the solvent system may be achieved throughmodification of the temperature and pressure of the solution, as idealdissolution conditions will vary by solvent selected and enhancersadded. Mechanical mixing methods employed may also vary by solvent typeand may vary from general agitation and mixing to ultrasonic disruptionof the protein aggregates. Additionally, the resultant dissolved silkconcentration may be tailored to range from about 1% (w/v) to about 30%(w/v). It may be possible to expand this range to include higherfractions of dissolved silk depending upon the specific solvent systemutilized. In one example, following initial dissolution of the processedsilk, the silk protein may be left in a pure aqueous solution at 8%(w/v) silk. This is accomplished by removal of the residual solventsystem while simultaneously ensuring that the aqueous component of thesilk solution is never fully removed nor compromised. In a situationwhich involves an initial solution of 200 g/L silk in a 9.3 M aqueoussolution of lithium bromide, this end is accomplished by a dialysisstep.

In aspects of this embodiment, the depolymerized silk fibroin (dissolvedsilk fibroin) has a concentration of, e.g., about 1%(w/v), about2%(w/v), about 3%(w/v), about 4%(w/v), about 5%(w/v), about 6%(w/v),about 7%(w/v), about 8%(w/v), about 9%(w/v), about 10%(w/v), about12%(w/v), about 15%(w/v), about 18%(w/v), about 20%(w/v), about25%(w/v), or about 30%(w/v). In other aspects of this embodiment, thedepolymerized silk fibroin (dissolved silk fibroin) has a concentrationof, e.g., at least 1%(w/v), at least 2%(w/v), at least 3%(w/v), at least4%(w/v), at least 5%(w/v), at least 6%(w/v), at least 7%(w/v), at least8%(w/v), at least 9%(w/v), at least 10%(w/v), at least 12%(w/v), atleast 15%(w/v), at least 18%(w/v), at least 20%(w/v), at least 25%(w/v),or at least 30%(w/v). In yet other aspects of this embodiment, thedepolymerized silk fibroin (dissolved silk fibroin) has a concentrationof, e.g., about 1%(w/v) to about 5% (w/v), about 1%(w/v) to about 10%(w/v), about 1%(w/v) to about 15% (w/v), about 1%(w/v) to about 20%(w/v), about 1%(w/v) to about 25% (w/v), about 1%(w/v) to about 30%(w/v), about 5%(w/v) to about 10% (w/v), about 5%(w/v) to about 15%(w/v), about 5%(w/v) to about 20% (w/v), about 5%(w/v) to about 25%(w/v), about 5%(w/v) to about 30% (w/v), about 10%(w/v) to about 15%(w/v), about 10%(w/v) to about 20% (w/v), about 10%(w/v) to about 25%(w/v), or about 10%(w/v) to about 30% (w/v).

Example dialysis conditions include a 3 mL-12 mL sample volume dialysiscassettes with 3.5 kD molecular weight cutoff cellulose membranesdialyzed for three days against ultra-pure water with a series of sixchanges at regular intervals while stirring constantly. Each cassette, 5mL-12 mL cartridge size, may be loaded (for example via 20-mL syringe)with 12 mL of a 20% solution of silk dissolved in 9.3 M lithium bromidevia an 18 gauge needle. The resultant silk solution may be 8%±0.5%(w/v). The silk solution may be stored at a range of −80° C. to 37° C.,such as 4° C. prior to use. One method is to dialyze the solutionagainst water using a 3.5 kD molecular weight cutoff cellulose membrane,for example, at one 12 mL cartridge per 1 L water in a 4 L beaker withstirring for 48 hours or 72 hours. Water may be changed several timesduring the dialysis, for example at 1 hour, 4 hours, 12 hours, 24 hours,and 36 hours (total of six rinses). In other embodiments, this membranemay take the shape of a cassette, tubing or any other semi-permeablemembrane in a batch, semi-continuous or continuous system. If desired,the concentration of silk in solution may be raised following theoriginal dialysis step by inclusion of a second dialysis against ahygroscopic polymer such as PEG, a poly(ethylene oxide) or amylase.

The parameters applied to the dialysis step may be altered according tothe specific needs or requirements of the particular solution systeminvolved. Although it may be undesirable to change membrane compositionor pore size in the interests of maintaining efficiency of the process,it would be possible to change the structuring of the dialysis barrier,as a dialysis tube or any large semi-permeable membrane of similarconstruction should suffice. Additionally it should be considered thatany alteration in the nature of the physical dialysis interface betweensolution and buffer might alter rates of ion flux and thereby createmembrane-localized boundary conditions which could affect solutiondialysis and gelation rate kinetics. The duration and volume ratiosassociated with this dialysis process must be tailored to any new systemas well, and removal of the solvent phase should be ensured afterpurification before proceeding.

It is also possible to change the buffer phase in the dialysis system,altering water purity or adding hygroscopic polymers to simultaneouslyremove ions and water from the initial silk solution. For example, ifnecessary, the silk solution can be concentrated by dialysis against ahygroscopic polymer, for example, PEG, a polyethylene oxide or amylase.The apparatus used for dialysis can be cassettes, tubing, or any othersemi-permeable membrane.

Insoluble debris may be removed from the dialyzed silk solution bycentrifugation or filtration. For example, the dialyzed silk may beremoved from the cassette with a needle and syringe (e.g., an 18 gneedle at 20 mL syringe), and placed into a clean centrifuge tube withsufficient volume (e.g., 40 mL). The centrifuge may be run at 30,000 grelative centrifugal force (RCF) for 30 minutes at 4° C. The resultingsupernatant may be collected and centrifuged again under identicalconditions, and the remaining supernatant collected (e.g., in a 50 mLtest tube) and stored at 4° C. The silk solution may also be evaluatedvia X-ray photoelectron spectroscopy (to check for lithium bromideresidue) and dry mass (to check solution for dry protein mass,concentration w/v).

Additionally, dependent upon the initial silk solvent, it might bedesirable to remove portions of either the silk phase or solvent phasefrom the solution via an affinity column separation. This could beuseful in either selectively binding specific solvent molecules orspecific solute molecules to be eluted later in a new solvent.

The possibility also exists for a lyophilization of the depolymerizedsilk fibroin (dissolved silk) followed by a reconstitution step. Thiswould be most useful in a case where removing a solvent, is unlikely toleave residue behind.

In the case of a lyophilized solution, either used as a purificationstep or as a procedure subsequent to purification, the type of solventused for reconstitution might be tailored for the process at hand.Desirable solvents might include but are not limited to aqueous alcoholsolutions, aqueous solutions with altered pH, and various organicsolutions. These solvents may be selected based upon a number ofparameters which may include but are not limited to an enhanced gelationrate, altered gel crystalline structure, altered solution intermediateshelf-life, altered silk solubility, and ability to interact withenvironmental milieu such as temperature and humidity.

In certain embodiments, a silk hydrogel is prepared from dissolved silkfibroin solution that uses an agent to enhance gelation and an agent toimprove the gel's biocompatibility. In some instances, the same agentboth enhances gelation and improves biocompatibility. An example agentthat both improves gel biocompatibility and serves as a gelationenhancer is an amphiphilic peptide which binds to silk molecules throughhydrophobic interactions, such as, e.g., a RGD motif containing peptidelike 23RGD. In other instances, different agents serve these purposes.An example of an agent that serves as a gelation enhancer is an alcohol,such as, e.g., ethanol, methanol, and isopropanol; glycerol; andacetone.

Regarding gelation enhancers, to accelerate the phenomenon of silkgelation, a depolymerized silk fibroin solution (dissolved silksolution) may be mixed with pure alcohol or aqueous alcohol solution atvaried volume ratios accompanied by mixing, either through stirring,shaking or any other form of agitation. This alcohol solution enhancermay then have a quantity of an amphiphilic peptide added as a furtherenhancer of the final gel outcome. The extent of acceleration may beheightened or lessened by adding a larger or smaller enhancer componentto the system.

In addition to organics, the gelation rate may be enhanced by increasingthe concentration of the depolymerized silk fibroin (dissolved silk).This is done by methods including but not limited to dialysis ofintermediate silk solution against a buffer incorporating a hygroscopicspecies such as polyethylene glycol, a lyophilization step, and anevaporation step. Increased temperature may also be used as an enhancerof the gelation process. In addition to this, manipulation ofintermediate silk solution pH by methods including but not limited todirect titration and gas exchange may be used to enhance the gelationprocess. Introduction of select ionic species including calcium andpotassium in particular may also be used to accelerate gelation rate.

Nucleating agents including organic and inorganic species, both solubleand insoluble in an aqueous silk solution intermediate may be used toenhance the gelation process. These may include but are not limited topeptide sequences which bind silk molecules, previously gelled silk, andpoorly soluble β-sheet rich structures. A further means of acceleratingthe gelation process is through the introduction of mechanicalexcitation. This might be imparted through a shearing device, ultrasounddevice, or mechanical mixer. It should be borne in mind that any ofthese factors might conceivably be used in concert with any other orgroup of others and that the regime would need to be tailored to thedesired outcome.

The time necessary for complete silk solution gelation may vary fromseconds to hours or days, depending on the values of the above mentionedparameters as well as the initial state of aggregation and organizationfound in the silk solution (FIG. 1). The volume fraction of addedenhancer may vary from about 0% to about 99% of the total system volume(i.e., either component may be added to a large excess of the other orin any relative concentration within the interval). The concentration ofsilk solution used can range from about 1% (w/v) to about 20% (w/v). Theenhancer can be added to silk solution or the silk solution can be addedto enhancer. The formed silk hydrogel may be further chemically orphysically cross-linked to gain altered mechanical properties.

In aspects of this embodiment, an enhancer solution is added to adepolymerized silk fibroin (dissolved silk fibroin) solution, thedepolymerized silk fibroin solution having a concentration ofdepolymerized silk fibroin of, e.g., about 1%(w/v), about 2%(w/v), about3%(w/v), about 4%(w/v), about 5%(w/v), about 6%(w/v), about 7%(w/v),about 8%(w/v), about 9%(w/v), about 10%(w/v), about 12%(w/v), about15%(w/v), about 18%(w/v), about 20%(w/v), about 25%(w/v), or about30%(w/v). In other aspects of this embodiment, an enhancer solution isadded to a depolymerized silk fibroin (dissolved silk fibroin) solution,the depolymerized silk fibroin solution having a concentration ofdepolymerized silk fibroin of, e.g., at least 1%(w/v), at least 2%(w/v),at least 3%(w/v), at least 4%(w/v), at least 5%(w/v), at least 6%(w/v),at least 7%(w/v), at least 8%(w/v), at least 9%(w/v), at least 10%(w/v),at least 12%(w/v), at least 15%(w/v), at least 18%(w/v), at least20%(w/v), at least 25%(w/v), or at least 30%(w/v). In yet other aspectsof this embodiment, an enhancer solution is added to a depolymerizedsilk fibroin (dissolved silk fibroin) solution, the depolymerized silkfibroin solution having a concentration of depolymerized silk fibroinof, e.g., about 1%(w/v) to about 5% (w/v), about 1%(w/v) to about 10%(w/v), about 1%(w/v) to about 15% (w/v), about 1%(w/v) to about 20%(w/v), about 1%(w/v) to about 25% (w/v), about 1%(w/v) to about 30%(w/v), about 5%(w/v) to about 10% (w/v), about 5%(w/v) to about 15%(w/v), about 5%(w/v) to about 20% (w/v), about 5%(w/v) to about 25%(w/v), about 5%(w/v) to about 30% (w/v), about 10%(w/v) to about 15%(w/v), about 10%(w/v) to about 20% (w/v), about 10%(w/v) to about 25%(w/v), or about 10%(w/v) to about 30% (w/v).

A further aspect of some embodiments relates to the inclusion of apeptide in the silk fibroin solution. Examples of such peptides includeamphiphilic peptides. Amphiphilic molecules possess both hydrophilic andhydrophobic properties. Many other amphiphilic molecules interactstrongly with biological membranes by insertion of the hydrophobic partinto the lipid membrane, while exposing the hydrophilic part to theaqueous environment. Particular embodiments of hydrogels include silkfibroin, silk fibroin with 23RGD, silk fibroin with alcohol and 23RGD,and silk fibroin with alcohol, 23RGD, and saline/PBS. The amount,relative ratio and sequence of adding the components will changeaccording to the specific requirement for the device.

Additionally, an amphiphilic peptide may accelerate the phenomenon ofsilk gelation under certain circumstances. Such gel may be producedthrough combination of dissolved silk fibroin solution and an enhancersolution of amphiphilic peptide in alcohol across the silk concentrationranges from about 1% (w/v) to about 20% (w/v), amphiphilic peptideconcentration ranges from about 1:100 to 100:1 moles 23RGD:moles silk,and alcohol concentration ranges from about 1% (v/v) to about 99% (v/v)before removal. Thus, for example, a particular silk gel is producedthrough direct contact between an aqueous solution of depolymerized silkfibroin and an enhancer solution comprising 23RGD in ethanol. Forexample, the dissolved silk solution may be mixed with a 23RGD suspendedin pure ethanol or aqueous ethanol solution at varied volume ratiosaccompanied by mixing, either through stirring, shaking or any otherform of agitation.

More specifically, as a non-limiting example, to infuse the silk fibroinhydrogel with 23RGD, the 23RGD is first dissolved in a solution ofethanol and water (e.g., 90% ethanol in purified water) in an amount togenerate the planned silk and 23RGD concentrations of the final gel, andmixed (e.g., vortexed until there is no visible 23RGD particulate). Thissolution is then mixed with dissolved silk solution (e.g., by pipettingrapidly for 1-2 seconds). The gelling mixture may be allowed to standcovered under ambient conditions for a suitable period, for example 24hours (or 24 hours after the gel has solidified depending on enhancerconditions).

The amount of time required for dissolved silk solutions to gel may varyfrom seconds to hours or days, depending on the ratio of silk solutionvolume and enhancer solution volume, dissolved silk fibroinconcentration, enhancer solution concentration, enhancer type andamphiphilic peptide concentration. The amphiphilic peptide may be mixedinto the dissolved silk solution in a variety of ways, for examplewater-dissolved amphiphilic peptide can be added to a dissolved silksolution to form a gel; an amphiphilic peptide can be added to water,blended with an alcohol, then added to a dissolved silk solution; or anamphiphilic peptide can be added to a silk fibroin hydrogel. The molarratio of amphiphilic peptide:silk fibroin can range from 100 to 0.01,the dissolved silk solution concentration can be from about 1% to about20%.

An example of an amphiphilic peptide is a 23RGD peptide having the aminoacid sequence:HOOC-Gly-Arg-Gly-Asp-Ile-Pro-Ala-Ser-Ser-Lys-Gly-Gly-Gly-Gly-Ser-Arg-Leu-Leu-Leu-Leu-Leu-Leu-Arg-NH₂(abbreviated HOOC-GRGDIPASSKG₄SRL₆R-NH₂) (SEQ ID NO: 1). Optionally,each of the arginine residues may be of the D-form, which may stabilizethe RG bond to serine proteases. Additionally, the COO-terminus may beacylated to block proteolysis. This example 23RGD has the amino acidsequence Ac-GdRGDIPASSKG₄SdRL_(6d)R-NH₂ (SEQ ID NO: 2). It may beadvantageous to include a spacer domain in the RGD peptide, for example,a peptide such as SG₄KSSAP (SEQ ID NO: 3) may present the RGD on thesurface of the silk biomaterial by optimally separating the cellattachment domain from the bonding sequence at the end of the peptide.The optional leucine tails of this example may interact in a fashionanalogous to a leucine zipper, and be driven by entropy from an aqueoussolution to form an approximation of a Langmuir-Blodgett (LB),monomolecular film on the surface of materials exposed to suchsolutions, thus presenting a ‘carpet’ of RGD attachment sites on thosesurfaces.

Other proteins or peptides may be used instead of 23RGD if such proteinsor peptides have the desired characteristics. Example characteristicsinclude hydrophilic domains that can interfere/enhance/affect silkgelation, and/or cell binding domains that enhance cell adhesion,spreading, and migration, such as RGD, KQAGDV (SEQ ID NO: 4), PHSRN (SEQID NO: 5), YIGSR (SEQ ID NO: 6), CDPGYIGSR (SEQ ID NO: 7), IKVAV (SEQ IDNO: 8), RNIAEIIKDI (SEQ ID NO: 9), YFQRYLI (SEQ ID NO: 10), PDSGR (SEQID NO: 11), FHRRIKA (SEQ ID NO: 12), PRRARV (SEQ ID NO: 13), andWQPPRARI (SEQ ID NO: 14). See Hersel et al., 24 Biomaterials 4285-415(2003).

In aspects of this embodiment, a hydrogel comprises a molar ratio ofamphiphilic peptide to silk fibroin of, e.g., about 100:1, about 90:1,about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1,about 20:1, about 10:1, about 7:1, about 5:1, about 3:1, about 1:1,about 1:3, about 1:5, about 1:7, about 1:10, about 1:20, about 1:30,about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, or about1:90, or about 1:100. In other aspects of this embodiment, a hydrogelcomprises a molar ratio of amphiphilic peptide to silk fibroin of, e.g.,at least 100:1, at least 90:1, at least 80:1, at least 70:1, at least60:1, at least 50:1, at least 40:1, at least 30:1, at least 20:1, atleast 10:1, at least 7:1, at least 5:1, at least 3:1, at least 1:1, atleast 1:3, at least 1:5, at least 1:7, at least 1:10, at least 1:20, atleast 1:30, at least 1:40, at least 1:50, at least 1:60, at least 1:70,at least 1:80, or at least 1:90, or at least 1:100. In yet other aspectsof this embodiment, a hydrogel comprises a molar ratio of amphiphilicpeptide to silk fibroin of, e.g., at most 100:1, at most 90:1, at most80:1, at most 70:1, at most 60:1, at most 50:1, at most 40:1, at most30:1, at most 20:1, at most 10:1, at most 7:1, at most 5:1, at most 3:1,at most 1:1, at most 1:3, at most 1:5, at most 1:7, at most 1:10, atmost 1:20, at most 1:30, at most 1:40, at most 1:50, at most 1:60, atmost 1:70, at most 1:80, or at most 1:90, or at most 1:100. In stillother aspects of this embodiment, a hydrogel comprises a molar ratio ofamphiphilic peptide to silk fibroin of, e.g., about 100:1 to about1:100; about 90:1 to about 1:90; about 80:1 to about 1:80; about 70:1 toabout 1:70; about 60:1 to about 1:60; about 50:1 to about 1:50; about40:1 to about 1:40; about 30:1 to about 1:30; about 20:1 to about 1:20;about 10:1 to about 1:10; about 7:1 to about 1:7; about 5:1 to about1:5; or about 3:1 to about 1:3.

The use of an amphiphilic peptide not only alters the protein structurecharacteristics of silk fibroin protein, but in so doing alters itsresistance to proteolytic bioresorption in vitro. These alterations inproteolytic bioresorption resistance stem from aspects of the proteinstructure alteration as α-helix and random coil are typically thought tobe less stable and therefore more susceptible to proteolyticbioresorption than β-sheet regions of silk. β-turn and β-strand regionsof the hydrogel disclosed in the present specification are mostresistant to proteolytic bioresorption as opposed to regions ofα-helixes and random coils. Through deliberate manipulation of thisprotein structure by means of controlled solution concentration andaddition of enhancer factors (type, concentration, and drivinggradient), gelation kinetics and resultant gel properties might becontrolled to deliver optimal outcomes in terms of degradative andresultant biological behaviors. The impact of amphiphilic peptideaddition to silk hydrogel in a silk hydrogel is evident upon examinationof data obtained through implantation studies conducted in vivo, bothsubcutaneously in rats and intradermally in the dermis of guinea pigs.See Example 10.

In an embodiment, a hydrogel comprises a silk fibroin protein whereinthe protein structure is resist bioresorption. In aspects of thisembodiment, a hydrogel comprising a silk fibroin has a protein structurethat makes the hydrogel resist to bioresorption for, e.g., about 10days, about 20 days, about 30 days, about 40 days, about 50 days, about60 days, about 70 days, about 80 days, or about 90 days. In otheraspects of this embodiment, a hydrogel comprising a silk fibroin has aprotein structure that makes the hydrogel resist to bioresorption for,e.g., at least 10 days, at least 20 days, at least 30 days, at least 40days, at least 50 days, at least 60 days, at least 70 days, at least 80days, or at least 90 days. In yet other aspects of this embodiment, ahydrogel comprising a silk fibroin has a protein structure that makesthe hydrogel resist to bioresorption for, e.g., about 10 days to about30 days, about 20 days to about 50 days, about 40 days to about 60 days,about 50 days to about 80 days, or about 60 days to about 90 days.

In another embodiment, a hydrogel comprising a silk fibroin and anamphiphilic peptide has a protein structure that makes the hydrogelresist to bioresorption. In aspects of this embodiment, a hydrogelcomprising a silk fibroin and an amphiphilic peptide has a proteinstructure that makes the hydrogel resist to bioresorption for, e.g.,about 10 days, about 20 days, about 30 days, about 40 days, about 50days, about 60 days, about 70 days, about 80 days, or about 90 days. Inother aspects of this embodiment, a hydrogel comprising a silk fibroinand an amphiphilic peptide has a protein structure that makes thehydrogel resist to bioresorption for, e.g., at least 10 days, at least20 days, at least 30 days, at least 40 days, at least 50 days, at least60 days, at least 70 days, at least 80 days, or at least 90 days. In yetother aspects of this embodiment, a hydrogel comprising a silk fibroinand an amphiphilic peptide has a protein structure that makes thehydrogel resist to bioresorption for, e.g., about 10 days to about 30days, about 20 days to about 50 days, about 40 days to about 60 days,about 50 days to about 80 days, or about 60 days to about 90 days.

In yet another embodiment, a hydrogel comprising a silk fibroin has aprotein structure that substantially includes β-turn and β-strandregions. In aspects of this embodiment, a hydrogel comprising a silkfibroin and an amphiphilic peptide has a protein structure including,e.g., about 10% β-turn and β-strand regions, about 20% β-turn andβ-strand regions, about 30% β-turn and β-strand regions, about 40%β-turn and β-strand regions, about 50% β-turn and β-strand regions,about 60% β-turn and β-strand regions, about 70% β-turn and β-strandregions, about 80% β-turn and β-strand regions, about 90% β-turn andβ-strand regions, or about 100% β-turn and β-strand regions. In otheraspects of this embodiment, a hydrogel comprising a silk fibroin has aprotein structure including, e.g., at least 10% β-turn and β-strandregions, at least 20% β-turn and β-strand regions, at least 30% β-turnand β-strand regions, at least 40% β-turn and β-strand regions, at least50% β-turn and β-strand regions, at least 60% β-turn and β-strandregions, at least 70% β-turn and β-strand regions, at least 80% β-turnand β-strand regions, at least 90% β-turn and β-strand regions, or atleast 95% β-turn and β-strand regions. In yet other aspects of thisembodiment, a hydrogel comprising a silk fibroin has a protein structureincluding, e.g., about 10% to about 30% β-turn and β-strand regions,about 20% to about 40% β-turn and β-strand regions, about 30% to about50% β-turn and β-strand regions, about 40% to about 60% β-turn andβ-strand regions, about 50% to about 70% β-turn and β-strand regions,about 60% to about 80% β-turn and β-strand regions, about 70% to about90% β-turn and β-strand regions, about 80% to about 100% β-turn andβ-strand regions, about 10% to about 40% β-turn and β-strand regions,about 30% to about 60% β-turn and β-strand regions, about 50% to about80% β-turn and β-strand regions, about 70% to about 100% β-turn andβ-strand regions, about 40% to about 80% β-turn and β-strand regions,about 50% to about 90% β-turn and β-strand regions, about 60% to about100% β-turn and β-strand regions, or about 50% to about 100% β-turn andβ-strand regions.

In yet another embodiment, a hydrogel comprising a silk fibroin and anamphiphilic peptide has a protein structure that is substantially-freeof α-helix and random coil regions. In aspects of this embodiment, ahydrogel comprising a silk fibroin has a protein structure including,e.g., about 5% α-helix and random coil regions, about 10% α-helix andrandom coil regions, about 15% α-helix and random coil regions, about20% α-helix and random coil regions, about 25% α-helix and random coilregions, about 30% α-helix and random coil regions, about 35% α-helixand random coil regions, about 40% α-helix and random coil regions,about 45% α-helix and random coil regions, or about 50% α-helix andrandom coil regions. In other aspects of this embodiment, a hydrogelcomprising a silk fibroin has a protein structure including, e.g., atmost 5% α-helix and random coil regions, at most 10% α-helix and randomcoil regions, at most 15% α-helix and random coil regions, at most 20%α-helix and random coil regions, at most 25% α-helix and random coilregions, at most 30% α-helix and random coil regions, at most 35%α-helix and random coil regions, at most 40% α-helix and random coilregions, at most 45% α-helix and random coil regions, or at most 50%α-helix and random coil regions. In yet other aspects of thisembodiment, a hydrogel comprising a silk fibroin has a protein structureincluding, e.g., about 5% to about 10% α-helix and random coil regions,about 5% to about 15% α-helix and random coil regions, about 5% to about20% α-helix and random coil regions, about 5% to about 25% α-helix andrandom coil regions, about 5% to about 30% α-helix and random coilregions, about 5% to about 40% α-helix and random coil regions, about 5%to about 50% α-helix and random coil regions, about 10% to about 20%α-helix and random coil regions, about 10% to about 30% α-helix andrandom coil regions, about 15% to about 25% α-helix and random coilregions, about 15% to about 30% α-helix and random coil regions, orabout 15% to about 35% α-helix and random coil regions.

In still another embodiment, a hydrogel comprising a silk fibroin and anamphiphilic peptide has a protein structure that substantially includesβ-turn and β-strand regions. In aspects of this embodiment, a hydrogelcomprising a silk fibroin and an amphiphilic peptide has a proteinstructure including, e.g., about 10% β-turn and β-strand regions, about20% β-turn and β-strand regions, about 30% β-turn and β-strand regions,about 40% β-turn and β-strand regions, about 50% β-turn and β-strandregions, about 60% β-turn and β-strand regions, about 70% β-turn andβ-strand regions, about 80% β-turn and β-strand regions, about 90%β-turn and β-strand regions, or about 100% β-turn and β-strand regions.In other aspects of this embodiment, a hydrogel comprising a silkfibroin and an amphiphilic peptide has a protein structure including,e.g., at least 10% β-turn and β-strand regions, at least 20% β-turn andβ-strand regions, at least 30% β-turn and β-strand regions, at least 40%β-turn and β-strand regions, at least 50% β-turn and β-strand regions,at least 60% β-turn and β-strand regions, at least 70% β-turn andβ-strand regions, at least 80% β-turn and β-strand regions, at least 90%β-turn and β-strand regions, or at least 95% β-turn and β-strandregions. In yet other aspects of this embodiment, a hydrogel comprisinga silk fibroin and an amphiphilic peptide has a protein structureincluding, e.g., about 10% to about 30% β-turn and β-strand regions,about 20% to about 40% β-turn and β-strand regions, about 30% to about50% β-turn and β-strand regions, about 40% to about 60% β-turn andβ-strand regions, about 50% to about 70% β-turn and β-strand regions,about 60% to about 80% β-turn and β-strand regions, about 70% to about90% β-turn and β-strand regions, about 80% to about 100% β-turn andβ-strand regions, about 10% to about 40% β-turn and β-strand regions,about 30% to about 60% β-turn and β-strand regions, about 50% to about80% β-turn and β-strand regions, about 70% to about 100% β-turn andβ-strand regions, about 40% to about 80% β-turn and β-strand regions,about 50% to about 90% β-turn and β-strand regions, about 60% to about100% β-turn and β-strand regions, or about 50% to about 100% β-turn andβ-strand regions.

In still another embodiment, a hydrogel comprising a silk fibroin and anamphiphilic peptide has a protein structure that is substantially-freeof α-helix and random coil regions. In aspects of this embodiment, ahydrogel comprising a silk fibroin and an amphiphilic peptide has aprotein structure including, e.g., about 5% α-helix and random coilregions, about 10% α-helix and random coil regions, about 15% α-helixand random coil regions, about 20% α-helix and random coil regions,about 25% α-helix and random coil regions, about 30% α-helix and randomcoil regions, about 35% α-helix and random coil regions, about 40%α-helix and random coil regions, about 45% α-helix and random coilregions, or about 50% α-helix and random coil regions. In other aspectsof this embodiment, a hydrogel comprising a silk fibroin and anamphiphilic peptide has a protein structure including, e.g., at most 5%α-helix and random coil regions, at most 10% α-helix and random coilregions, at most 15% α-helix and random coil regions, at most 20%α-helix and random coil regions, at most 25% α-helix and random coilregions, at most 30% α-helix and random coil regions, at most 35%α-helix and random coil regions, at most 40% α-helix and random coilregions, at most 45% α-helix and random coil regions, or at most 50%α-helix and random coil regions. In yet other aspects of thisembodiment, a hydrogel comprising a silk fibroin and an amphiphilicpeptide has a protein structure including, e.g., about 5% to about 10%α-helix and random coil regions, about 5% to about 15% α-helix andrandom coil regions, about 5% to about 20% α-helix and random coilregions, about 5% to about 25% α-helix and random coil regions, about 5%to about 30% α-helix and random coil regions, about 5% to about 40%α-helix and random coil regions, about 5% to about 50% α-helix andrandom coil regions, about 10% to about 20% α-helix and random coilregions, about 10% to about 30% α-helix and random coil regions, about15% to about 25% α-helix and random coil regions, about 15% to about 30%α-helix and random coil regions, or about 15% to about 35% α-helix andrandom coil regions.

Aspects of the present specification provide, in part, a silk fibroinhydrogel having a hardness. Hardness refers to various properties of anobject in the solid phase that gives it high resistance to various kindsof shape change when force is applied. Hardness is measured using adurometer and is a unitless value that ranges from zero to 100. Theability or inability of a hydrogel to be easily compressed will affectits suitability for application in different tissue replacement roles,i.e., mechanical compliance as bone, fat, connective tissue. Hardnesswill also affect the ability of a hydrogel to be effectively comminuted,the reason being that a hard material may be more easily andconsistently comminuted. Hardness will also affect extrudability, as asoft material may be more readily able to be slightly compressed duringinjection to pack with other particles or change shape to pass through asyringe barrel or needle.

In an embodiment, a silk fibroin hydrogel exhibits low hardness. Inaspects of this embodiment, a silk fibroin hydrogel exhibits a hardnessof, e.g., about 5, about 10, about 15, about 20, about 25, about 30, orabout 35. In other aspects of this embodiment, a silk fibroin hydrogelexhibits a hardness of, e.g., at most 5, at most 10, at most 15, at most20, at most 25, at most 30, or at most 35. In yet other aspects of thisembodiment, a silk fibroin hydrogel exhibits a hardness of, e.g., about5 to about 35, about 10 to about 35, about 15 to about 35, about 20 toabout 35, or about 25 to about 35, about 5 to about 40, about 10 toabout 40, about 15 to about 40, about 20 to about 40, about 25 to about40, or about 30 to about 40.

In an embodiment, a silk fibroin hydrogel exhibits medium hardness. Inaspects of this embodiment, a silk fibroin hydrogel exhibits a hardnessof, e.g., about 40, about 45, about 50, about 55, or about 60. In otheraspects of this embodiment, a silk fibroin hydrogel exhibits a hardnessof, e.g., at least 40, at least 45, at least 50, at least 55, or atleast 60. In yet other aspects of this embodiment, a silk fibroinhydrogel exhibits a hardness of, e.g., at most 40, at most 45, at most50, at most 55, or at most 60. In still other aspects of thisembodiment, a silk fibroin hydrogel exhibits a hardness of, e.g., about35 to about 60, about 35 to about 55, about 35 to about 50, about 35 toabout 45, about 40 to about 60, about 45 to about 60, about 50 to about60, about 55 to about 60, about 40 to about 65, about 45 to about 65,about 50 to about 65, about 55 to about 65.

In another embodiment, a silk fibroin hydrogel exhibits high hardness.In aspects of this embodiment, a silk fibroin hydrogel exhibits ahardness of, e.g., about 65, about 70, about 75, about 80, about 85,about 90, about 95, or about 100. In other aspects of this embodiment, asilk fibroin hydrogel exhibits a hardness of, e.g., at least 65, atleast 70, at least 75, at least 80, at least 85, at least 90, at least95, or at least 100. In yet other aspects of this embodiment, a silkfibroin hydrogel exhibits a hardness of, e.g., about 65 to about 100,about 70 to about 100, about 75 to about 100, about 80 to about 100,about 85 to about 100, about 90 to about 100, about 65 to about 75,about 65 to about 80, about 65 to about 85, about 65 to about 90, about65 to about 95, about 60 to about 75, about 60 to about 80, about 60 toabout 85, about 60 to about 90, or about 60 to about 95.

In an embodiment, a silk fibroin hydrogel exhibits high resistant todeformation. In aspects of this embodiment, a silk fibroin hydrogelexhibits resistant to deformation of, e.g., about 100%, about 99%, about98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%,about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, orabout 85%. In other aspects of this embodiment, a silk fibroin hydrogelexhibits resistant to deformation of, e.g., at least 99%, at least 98%,at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, atleast 92%, at least 91%, at least 90%, at least 89%, at least 88%, atleast 87%, at least 86%, or at least 85%. In yet other aspects of thisembodiment, a silk fibroin hydrogel exhibits resistant to deformationof, e.g., at most 99%, at most 98%, at most 97%, at most 96%, at most95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, atmost 89%, at most 88%, at most 87%, at most 86%, or at most 85%. Instill aspects of this embodiment, a silk fibroin hydrogel exhibitsresistant to deformation of, e.g., about 85% to about 100%, about 87% toabout 100%, about 90% to about 100%, about 93% to about 100%, about 95%to about 100%, or about 97% to about 100%.

A silk fibroin hydrogel exhibits an elastic modulus. Elastic modulus, ormodulus of elasticity, refers to the ability of a hydrogel material toresists deformation, or, conversely, an object's tendency to benon-permanently deformed when a force is applied to it. The elasticmodulus of an object is defined as the slope of its stress-strain curvein the elastic deformation region: λ=stress/strain, where λ is theelastic modulus in Pascal's; stress is the force causing the deformationdivided by the area to which the force is applied; and strain is theratio of the change caused by the stress to the original state of theobject. Specifying how stresses are to be measured, includingdirections, allows for many types of elastic moduli to be defined. Thethree primary elastic moduli are tensile modulus, shear modulus, andbulk modulus.

Tensile modulus (E) or Young's modulus is an objects response to linearstrain, or the tendency of an object to deform along an axis whenopposing forces are applied along that axis. It is defined as the ratioof tensile stress to tensile strain. It is often referred to simply asthe elastic modulus. The shear modulus or modulus of rigidity refers toan object's tendency to shear (the deformation of shape at constantvolume) when acted upon by opposing forces. It is defined as shearstress over shear strain. The shear modulus is part of the derivation ofviscosity. The shear modulus is concerned with the deformation of asolid when it experiences a force parallel to one of its surfaces whileits opposite face experiences an opposing force (such as friction). Thebulk modulus (K) describes volumetric elasticity or an object'sresistance to uniform compression, and is the tendency of an object todeform in all directions when uniformly loaded in all directions. It isdefined as volumetric stress over volumetric strain, and is the inverseof compressibility. The bulk modulus is an extension of Young's modulusto three dimensions.

In another embodiment, a silk fibroin hydrogel exhibits a tensilemodulus. In aspects of this embodiment, a silk fibroin hydrogel exhibitsa tensile modulus of, e.g., about 1 MPa, about 10 MPa, about 20 MPa,about 30 MPa, about 40 MPa, about 50 MPa, about 60 MPa, about 70 MPa,about 80 MPa, about 90 MPa, about 100 MPa, about 200 MPa, about 300 MPa,about 400 MPa, about 500 MPa, about 750 MPa, about 1 GPa, about 5 GPa,about 10 GPa, about 15 GPa, about 20 GPa, about 25 GPa, or about 30 GPa.In other aspects of this embodiment, a silk fibroin hydrogel exhibits atensile modulus of, e.g., at least 1 MPa, at least 10 MPa, at least 20MPa, at least 30 MPa, at least 40 MPa, at least 50 MPa, at least 60 MPa,at least 70 MPa, at least 80 MPa, at least 90 MPa, at least 100 MPa, atleast 200 MPa, at least 300 MPa, at least 400 MPa, at least 500 MPa, atleast 750 MPa, at least 1 GPa, at least 5 GPa, at least 10 GPa, at least15 GPa, at least 20 GPa, at least 25 GPa, or at least 30 GPa In yetother aspects of this embodiment, a silk fibroin hydrogel exhibits atensile modulus of, e.g., about 1 MPa to about 30 MPa, about 10 MPa toabout 50 MPa, about 25 MPa to about 75 MPa, about 50 MPa to about 100MPa, about 100 MPa to about 300 MPa, about 200 MPa to about 400 MPa,about 300 MPa to about 500 MPa, about 100 MPa to about 500 MPa, about250 MPa to about 750 MPa, about 500 MPa to about 1 GPa, about 1 GPa toabout 30 GPa, about 10 GPa to about 30 GPa.

In another embodiment, a silk fibroin hydrogel exhibits shear modulus.In aspects of this embodiment, a silk fibroin hydrogel exhibits a shearmodulus of, e.g., about 1 MPa, about 10 MPa, about 20 MPa, about 30 MPa,about 40 MPa, about 50 MPa, about 60 MPa, about 70 MPa, about 80 MPa,about 90 MPa, about 100 MPa, about 200 MPa, about 300 MPa, about 400MPa, about 500 MPa, about 750 MPa, about 1 GPa, about 5 GPa, about 10GPa, about 15 GPa, about 20 GPa, about 25 GPa, or about 30 GPa. In otheraspects of this embodiment, a silk fibroin hydrogel exhibits a shearmodulus of, e.g., at least 1 MPa, at least 10 MPa, at least 20 MPa, atleast 30 MPa, at least 40 MPa, at least 50 MPa, at least 60 MPa, atleast 70 MPa, at least 80 MPa, at least 90 MPa, at least 100 MPa, atleast 200 MPa, at least 300 MPa, at least 400 MPa, at least 500 MPa, atleast 750 MPa, at least 1 GPa, at least 5 GPa, at least 10 GPa, at least15 GPa, at least 20 GPa, at least 25 GPa, or at least 30 GPa In yetother aspects of this embodiment, a silk fibroin hydrogel exhibits ashear modulus of, e.g., about 1 MPa to about 30 MPa, about 10 MPa toabout 50 MPa, about 25 MPa to about 75 MPa, about 50 MPa to about 100MPa, about 100 MPa to about 300 MPa, about 200 MPa to about 400 MPa,about 300 MPa to about 500 MPa, about 100 MPa to about 500 MPa, about250 MPa to about 750 MPa, about 500 MPa to about 1 GPa, about 1 GPa toabout 30 GPa, about 10 GPa to about 30 GPa.

In another embodiment, a silk fibroin hydrogel exhibits a bulk modulus.In aspects of this embodiment, a silk fibroin hydrogel exhibits a bulkmodulus of, e.g., about 5 GPa, about 6 GPa, about 7 GPa, about 8 GPa,about 9 GPa, about 10 GPa, about 15 GPa, about 20 GPa, about 25 GPa,about 30 GPa, about 35 GPa, about 40 GPa, about 45 GPa, about 50 GPa,about 60 GPa, about 70 GPa, about 80 GPa, about 90 GPa, about 100 GPa.In other aspects of this embodiment, a silk fibroin hydrogel exhibits abulk modulus of, e.g., at least 5 GPa, at least 6 GPa, at least 7 GPa,at least 8 GPa, at least 9 GPa, at least 10 GPa, at least 15 GPa, atleast 20 GPa, at least 25 GPa, at least 30 GPa, at least 35 GPa, atleast 40 GPa, at least 45 GPa, at least 50 GPa, at least 60 GPa, atleast 70 GPa, at least 80 GPa, at least 90 GPa, at least 100 GPa. In yetother aspects of this embodiment, a silk fibroin hydrogel exhibits abulk modulus of, e.g., about 5 GPa to about 50 GPa, about 5 GPa to about100 GPa, about 10 GPa to about 50 GPa, about 10 GPa to about 100 GPa, orabout 50 GPa to about 100 GPa.

A silk fibroin hydrogel exhibits high tensile strength. Tensile strengthhas three different definitional points of stress maxima. Yield strengthrefers to the stress at which material strain changes from elasticdeformation to plastic deformation, causing it to deform permanently.Ultimate strength refers to the maximum stress a material can withstandwhen subjected to tension, compression or shearing. It is the maximumstress on the stress-strain curve. Breaking strength refers to thestress coordinate on the stress-strain curve at the point of rupture, orwhen the material pulls apart.

In another embodiment, a silk fibroin hydrogel exhibits high yieldstrength relative to other polymer classes. In aspects of thisembodiment, an elastomer matrix defining an array of interconnectedpores exhibits a yield strength of, e.g., about 0.1 MPa, about 0.5 MPa,about 1 MPa, about 5 MPa, about 10 MPa, about 20 MPa, about 30 MPa,about 40 MPa, about 50 MPa, about 60 MPa, about 70 MPa, about 80 MPa,about 90 MPa, about 100 MPa, about 200 MPa, about 300 MPa, about 400MPa, about 500 MPa. In other aspects of this embodiment, a silk fibroinhydrogel exhibits a yield strength of, e.g., at least 0.1 MPa, at least0.5 MPa, at least 1 MPa, at least 5 MPa, at least 10 MPa, at least 20MPa, at least 30 MPa, at least 40 MPa, at least 50 MPa, at least 60 MPa,at least 70 MPa, at least 80 MPa, at least 90 MPa, at least 100 MPa, atleast 200 MPa, at least 300 MPa, at least 400 MPa, at least 500 MPa. Inyet other aspects of this embodiment, a silk fibroin hydrogel exhibits ayield strength of, e.g., at most 1 MPa, at most 5 MPa, at most 10 MPa,at most 20 MPa, at most 30 MPa, at most 40 MPa, at most 50 MPa, at most60 MPa, at most 70 MPa, at most 80 MPa, at most 90 MPa, at most 100 MPa,at most 200 MPa, at most 300 MPa, at most 400 MPa, at most 500 MPa, atmost 600 MPa, at most 700 MPa, at most 800 MPa, at most 900 MPa, at most1000 MPa, at most 1500 MPa, or at most 2000 MPa. In still other aspectsof this embodiment, a silk fibroin hydrogel exhibits a yield strengthof, e.g., about 1 MPa to about 50 MPa, about 1 MPa to about 60 MPa,about 1 MPa to about 70 MPa, about 1 MPa to about 80 MPa, about 1 MPa toabout 90 MPa, about 1 MPa to about 100 MPa, about 10 MPa to about 50MPa, about 10 MPa to about 60 MPa, about 10 MPa to about 70 MPa, about10 MPa to about 80 MPa, about 10 MPa to about 90 MPa, about 10 MPa toabout 100 MPa, about 10 MPa to about 200 MPa, about 10 MPa to about 300MPa, or about 100 MPa to about 300 MPa.

In another embodiment, a silk fibroin hydrogel exhibits high ultimatestrength. In aspects of this embodiment, a silk fibroin hydrogelexhibits an ultimate strength of, e.g., about 0.1 MPa, about 0.5 MPa,about 1 MPa, about 5 MPa, about 10 MPa, about 20 MPa, about 30 MPa,about 40 MPa, about 50 MPa, about 60 MPa, about 70 MPa, about 80 MPa,about 90 MPa, about 100 MPa, about 200 MPa, about 300 MPa, about 400MPa, about 500 MPa. In other aspects of this embodiment, a silk fibroinhydrogel exhibits an ultimate strength of, e.g., at least 0.1 MPa, atleast 0.5 MPa, at least 1 MPa, at least 5 MPa, at least 10 MPa, at least20 MPa, at least 30 MPa, at least 40 MPa, at least 50 MPa, at least 60MPa, at least 70 MPa, at least 80 MPa, at least 90 MPa, at least 100MPa, at least 200 MPa, at least 300 MPa, at least 400 MPa, at least 500MPa. In yet other aspects of this embodiment, a silk fibroin hydrogelexhibits an ultimate strength of, e.g., at most 1 MPa, at most 5 MPa, atmost 10 MPa, at most 20 MPa, at most 30 MPa, at most 40 MPa, at most 50MPa, at most 60 MPa, at most 70 MPa, at most 80 MPa, at most 90 MPa, atmost 100 MPa, at most 200 MPa, at most 300 MPa, at most 400 MPa, at most500 MPa, at most 600 MPa, at most 700 MPa, at most 800 MPa, at most 900MPa, at most 1000 MPa, at most 1500 MPa, or at most 2000 MPa. In stillother aspects of this embodiment, a silk fibroin hydrogel exhibits anultimate strength of, e.g., about 1 MPa to about 50 MPa, about 1 MPa toabout 60 MPa, about 1 MPa to about 70 MPa, about 1 MPa to about 80 MPa,about 1 MPa to about 90 MPa, about 1 MPa to about 100 MPa, about 10 MPato about 50 MPa, about 10 MPa to about 60 MPa, about 10 MPa to about 70MPa, about 10 MPa to about 80 MPa, about 10 MPa to about 90 MPa, about10 MPa to about 100 MPa, about 10 MPa to about 200 MPa, about 10 MPa toabout 300 MPa, or about 100 MPa to about 300 MPa.

In another embodiment, a silk fibroin hydrogel exhibits high breakingstrength. In aspects of this embodiment, a silk fibroin hydrogelexhibits a breaking strength of, e.g., about 0.1 MPa, about 0.5 MPa,about 1 MPa, about 5 MPa, about 10 MPa, about 20 MPa, about 30 MPa,about 40 MPa, about 50 MPa, about 60 MPa, about 70 MPa, about 80 MPa,about 90 MPa, about 100 MPa, about 200 MPa, about 300 MPa, about 400MPa, about 500 MPa. In other aspects of this embodiment, a silk fibroinhydrogel exhibits a breaking strength of, e.g., at least 0.1 MPa, atleast 0.5 MPa, at least 1 MPa, at least 5 MPa, at least 10 MPa, at least20 MPa, at least 30 MPa, at least 40 MPa, at least 50 MPa, at least 60MPa, at least 70 MPa, at least 80 MPa, at least 90 MPa, at least 100MPa, at least 200 MPa, at least 300 MPa, at least 400 MPa, at least 500MPa. In yet other aspects of this embodiment, a silk fibroin hydrogelexhibits a breaking strength of, e.g., at most 1 MPa, at most 5 MPa, atmost 10 MPa, at most 20 MPa, at most 30 MPa, at most 40 MPa, at most 50MPa, at most 60 MPa, at most 70 MPa, at most 80 MPa, at most 90 MPa, atmost 100 MPa, at most 200 MPa, at most 300 MPa, at most 400 MPa, at most500 MPa, at most 600 MPa, at most 700 MPa, at most 800 MPa, at most 900MPa, at most 1000 MPa, at most 1500 MPa, or at most 2000 MPa. In stillother aspects of this embodiment, a silk fibroin hydrogel exhibits abreaking strength of, e.g., about 1 MPa to about 50 MPa, about 1 MPa toabout 60 MPa, about 1 MPa to about 70 MPa, about 1 MPa to about 80 MPa,about 1 MPa to about 90 MPa, about 1 MPa to about 100 MPa, about 10 MPato about 50 MPa, about 10 MPa to about 60 MPa, about 10 MPa to about 70MPa, about 10 MPa to about 80 MPa, about 10 MPa to about 90 MPa, about10 MPa to about 100 MPa, about 10 MPa to about 200 MPa, about 10 MPa toabout 300 MPa, or about 100 MPa to about 300 MPa.

Aspects of the present specification provide, in part, a silk fibroinhydrogel having a transparency and/or translucency. Transparency (alsocalled pellucidity or diaphaneity) is the physical property of allowinglight to pass through a material, whereas translucency (also calledtranslucence or translucidity) only allows light to pass throughdiffusely. The opposite property is opacity. Transparent materials areclear, while translucent ones cannot be seen through clearly. The silkfibroin hydrogels disclosed in the present specification may, or maynot, exhibit optical properties such as transparency and translucency.In certain cases, e.g., superficial line filling, it would be anadvantage to have an opaque hydrogel. In other cases such as developmentof a lens or a “humor” for filling the eye, it would be an advantage tohave a translucent hydrogel. These properties could be modified byaffecting the structural distribution of the hydrogel material. Factorsused to control a hydrogels optical properties include, withoutlimitation, silk fibroin concentration, gel crystallinity, and hydrogelhomogeneity.

When light encounters a material, it can interact with it in severaldifferent ways. These interactions depend on the nature of the light(its wavelength, frequency, energy, etc.) and the nature of thematerial. Light waves interact with an object by some combination ofreflection, and transmittance with refraction. As such, an opticallytransparent material allows much of the light that falls on it to betransmitted, with little light being reflected. Materials which do notallow the transmission of light are called optically opaque or simplyopaque.

In an embodiment, a silk fibroin hydrogel is optically transparent. Inaspects of this embodiment, a silk fibroin hydrogel transmits, e.g.,about 75% of the light, about 80% of the light, about 85% of the light,about 90% of the light, about 95% of the light, or about 100% of thelight. In other aspects of this embodiment, a silk fibroin hydrogeltransmits, e.g., at least 75% of the light, at least 80% of the light,at least 85% of the light, at least 90% of the light, or at least 95% ofthe light. In yet other aspects of this embodiment, a silk fibroinhydrogel transmits, e.g., about 75% to about 100% of the light, about80% to about 100% of the light, about 85% to about 100% of the light,about 90% to about 100% of the light, or about 95% to about 100% of thelight.

In another embodiment, a silk fibroin hydrogel is optically opaque. Inaspects of this embodiment, a silk fibroin hydrogel transmits, e.g.,about 5% of the light, about 10% of the light, about 15% of the light,about 20% of the light, about 25% of the light, about 30% of the light,about 35% of the light, about 40% of the light, about 45% of the light,about 50% of the light, about 55% of the light, about 60% of the light,about 65% of the light, or about 70% of the light. In other aspects ofthis embodiment, a silk fibroin hydrogel transmits, e.g., at most 5% ofthe light, at most 10% of the light, at most 15% of the light, at most20% of the light, at most 25% of the light, at most 30% of the light, atmost 35% of the light, at most 40% of the light, at most 45% of thelight, at most 50% of the light, at most 55% of the light, at most 60%of the light, at most 65% of the light, at most 70% of the light, or atmost 75% of the light. In other aspects of this embodiment, a silkfibroin hydrogel transmits, e.g., about 5% to about 15%, about 5% toabout 20%, about 5% to about 25%, about 5% to about 30%, about 5% toabout 35%, about 5% to about 40%, about 5% to about 45%, about 5% toabout 50%, about 5% to about 55%, about 5% to about 60%, about 5% toabout 65%, about 5% to about 70%, about 5% to about 75%, about 15% toabout 20%, about 15% to about 25%, about 15% to about 30%, about 15% toabout 35%, about 15% to about 40%, about 15% to about 45%, about 15% toabout 50%, about 15% to about 55%, about 15% to about 60%, about 15% toabout 65%, about 15% to about 70%, about 15% to about 75%, about 25% toabout 35%, about 25% to about 40%, about 25% to about 45%, about 25% toabout 50%, about 25% to about 55%, about 25% to about 60%, about 25% toabout 65%, about 25% to about 70%, or about 25% to about 75%.

In an embodiment, a silk fibroin hydrogel is optically translucent. Inaspects of this embodiment, a silk fibroin hydrogel diffusely transmits,e.g., about 75% of the light, about 80% of the light, about 85% of thelight, about 90% of the light, about 95% of the light, or about 100% ofthe light. In other aspects of this embodiment, a silk fibroin hydrogeldiffusely transmits, e.g., at least 75% of the light, at least 80% ofthe light, at least 85% of the light, at least 90% of the light, or atleast 95% of the light. In yet other aspects of this embodiment, a silkfibroin hydrogel diffusely transmits, e.g., about 75% to about 100% ofthe light, about 80% to about 100% of the light, about 85% to about 100%of the light, about 90% to about 100% of the light, or about 95% toabout 100% of the light.

To remove enhancer species from the formed gel and become a morecomplete hydrogel, the formed gel may be leeched against water, forexample under ambient temperature and pressure conditions for three dayswith five changes of water. The gel may be leeched against ultra-purewater of a volume at least 100-times that of the gel. More specifically,for example, the gels may be placed in a bulk of purified water and therinse changed at hours 12, 24 and 48 with 15 mL gel per 1.5 L water. Thenumber of rinses and volume ratios involved may be altered so long asthe resultant hydrogel is substantially free of residual gelationenhancer.

The hydrogel may then be further processed for cleaning, loading, andsterilizing for use. For example, the hydrogel may be pulverized andmixed with saline solution. In a particular example, the gel may bemilled to a particle size from about 10 μm to about 1000 μm in diameter,such as 15 μm to 30 μm. Saline is then added to the hydrogel as acarrier phase by first determining the volume of a bulk of hydrogel,then vigorously pulverizing the hydrogel while incorporating anappropriate volume of saline to the hydrogel to achieve a desiredcarrier to hydrogel ratio. For example, hydrogel milling may beaccomplished in one example by means of a forced sieving of bulkhydrogel material through a series of stainless steel cloth sieves ofdecreasing pore sizes. In another example, hydrogel may be loaded into asyringe and pulverized with a spatula to a fine paste with saline. Thepresent hydrogel formulations are preferably sterile.

In an aspect of this embodiment, a hydrogel formulation comprises a gelphase including hydrogel particles having a particle size from about 5μm² to about 1000 μm² in cross-sectional area. In aspects of thisembodiment, a hydrogel formulation comprises a gel phase includinghydrogel particles having a mean particle size of, e.g., about 5 μm²,about 10 μm², about 15 μm², about 20 μm², about 25 μm², about 30 μm²,about 35 μm², about 40 μm², about 45 μm², about 50 μm², about 60 μm²,about 70 μm², about 80 μm², about 90 μm², about 100 μm², about 200 μm²,about 300 μm², about 400 μm², about 500 μm², about 600 μm², about 700μm², about 800 μm², about 900 μm², or about 1000 μm² in cross-sectionalarea. In other aspects of the embodiment, a hydrogel formulationcomprises a gel phase including hydrogel particles having a meanparticle size of, e.g., at least 5 μm², at least 10 μm², at least 15μm², at least 20 μm², at least 25 μm², at least 30 μm², at least 35 μm²,at least 40 μm², at least 45 μm², at least 50 μm², at least 60 μm², atleast 70 μm², at least 80 μm², at least 90 μm², at least 100 μm², atleast 200 μm², at least 300 μm², at least 400 μm², at least 500 μm², atleast 600 μm², at least 700 μm², at least 800 μm², at least 900 μm², orat least 1000 μm² in cross-sectional area. In yet other aspects of thisembodiment, a hydrogel formulation comprises a gel phase includinghydrogel particles having a mean particle size of, e.g., at most 5 μm²,at most 10 μm², at most 15 μm², at most 20 μm², at most 25 μm², at most30 μm², at most 35 μm², at most 40 μm², at most 45 μm², at most 50 μm²,at most 60 μm², at most 70 μm², at most 80 μm², at most 90 μm², at most100 μm², at most 200 μm², at most 300 μm², at most 400 μm², at most 500μm², at most 600 μm², at most 700 μm², at most 800 μm², at most 900 μm²,or at most 1000 μm² in cross-sectional area. In still other aspects ofthe embodiment, a hydrogel formulation comprises a gel phase includinghydrogel particles having a mean particle size of, e.g., about 5 μm² toabout 50 μm², about 25 μm² to about 75 μm², about 50 μm² to about 100μm², about 100 μm² to about 300 μm², about 200 μm² to about 400 μm²,about 300 μm² to about 500 μm², about 400 μm² to about 600 μm², about500 μm² to about 700 μm², about 600 μm² to about 800 μm², about 700 μm²to about 900 μm², about 800 μm² to about 1000 μm², about 100 μm² toabout 400 μm², about 300 μm² to about 600 μm², about 500 μm² to about800 μm², or about 700 μm² to about 1000 μm², in cross-sectional area.

In aspects of this embodiment, a hydrogel formulation comprises a gelphase including hydrogel particles having a particle size of, e.g., atleast 5 μm², at least 10 μm², at least 15 μm², at least 20 μm², at least25 μm², at least 30 μm², at least 35 μm², at least 40 μm², at least 45μm², at least 50 μm², at least 60 μm², at least 70 μm², at least 80 μm²,at least 90 μm², at least 100 μm², at least 200 μm², at least 300 μm²,at least 400 μm², at least 500 μm², at least 600 μm², at least 700 μm²,at least 800 μm², at least 900 μm², or at least 1000 μm² incross-sectional area. In other aspects of this embodiment, a hydrogelformulation comprises a gel phase including hydrogel particles having aparticle size of, e.g., at most 5 μm², at most 10 μm², at most 15 μm²,at most 20 μm², at most 25 μm², at most 30 μm², at most 35 μm², at most40 μm², at most 45 μm², at most 50 μm², at most 60 μm², at most 70 μm²,at most 80 μm², at most 90 μm², at most 100 μm², at most 200 μm², atmost 300 μm², at most 400 μm², at most 500 μm², at most 600 μm², at most700 μm², at most 800 μm², at most 900 μm², or at most 1000 μm² incross-sectional area. In yet other aspects of the embodiment, a hydrogelformulation comprises a gel phase including hydrogel particles having aparticle size of, e.g., about 5 μm² to about 50 μm², about 25 μm² toabout 75 μm², about 50 μm² to about 100 μm², about 100 μm² to about 300μm², about 200 μm² to about 400 μm², about 300 μm² to about 500 μm²,about 400 μm² to about 600 μm², about 500 μm² to about 700 μm², about600 μm² to about 800 μm², about 700 μm² to about 900 μm², about 800 μm²to about 1000 μm², about 100 μm² to about 400 μm², about 300 μm² toabout 600 μm², about 500 μm² to about 800 μm², or about 700 μm² to about1000 μm², in cross-sectional area.

Aspects of the present hydrogel formulations provide, in part, a carrierphase. A carrier phase is advantageously a physiologically-acceptablecarrier phase and may include one or more conventional excipients usefulin pharmaceutical compositions. As used herein, the term “aphysiologically-acceptable carrier phase” refers to a carrier phase inaccord with, or characteristic of, the normal functioning of a livingorganism. As such, administration of a hydrogel formulation disclosed inthe present composition comprises a carrier phase that has substantiallyno long term or permanent detrimental effect when administered tomammal. The present compositions preferably include a carrier phasewhere a major of the volume is water or saline. However, other usefulcarrier phases include any physiologically tolerable buffer, serum orother protein solutions.

The volume of carrier phase per volume of gel phase may be increased ordecreased in a range between 0% to 100% depending upon the desiredphysical properties of the resultant formulation including dosedelivery, viscosity, injectability, and desired in vivo behavioralcharacteristics. This formulation is then mixed until achieving a“uniform” hydrogel formulation consistency which may be termed anemulsion or suspension. More specifically, for example, a hydrogel maybe passed through an 18 g needle several times to decrease particlesize, injecting back and forth between a pair of syringes, then thisprocedure repeated with 22 g needles affixed to 1 mL syringes.Advantages derived from adding a carrier phase to the gel phase includedecreased viscosity in the extracellular in vivo microenvironment;release of local mechanical stress loading after hydrogel formulationadministration; and improved ionic composition resulting in improvedbiocompatibility.

The silk hydrogel disclosed in the present specification may beformulated using material processing constraints such as silkconcentration and saline concentration to tailor material longevity invivo. In one example, a silk gel might be tailored for a persistence offive weeks to six weeks in vivo by using a 1%-3% (w/v) silk gel with25%-50% (v/v) saline carrier. In another example, a silk gel might betailored for a persistence of two months to three months in vivo byusing a 3%-5% (w/v) silk gel with 20%-40% (v/v) saline. In anotherexample, a silk gel might be tailored for a persistence of 5-6 months byusing 4-6% (w/v) silk gel with 20-40% (v/v) saline. In another example,a silk gel might be tailored for a persistence of 7-10 months by using a6-8% (w/v) silk gel with 20-30% (v/v) saline. The persistence of thesematerials might also be increased or decreased by increasing ordecreasing particle size respectively.

Gel emulsion saline content and gel silk concentration could be used tomodify the mechanical profile of the silk gel materials for particularapplications. For example, a gel emulsion of about 1% (w/v) to about 5%(w/v) silk gel concentration with 5%-95% lubricant (e.g., 5%-95% (w/v)saline/PBS) may be useful as a dermal filler, bulking agent, camouflageagent, intramuscular or sub-Q filler, or pharmaceutical delivery vector.A gel emulsion of, for example, about 5% (w/v) to about 8% (w/v) silkgel concentration with 0% to about 30% lubricant fluid may be useful inbone defects or cartilage defects.

Aspects of the present specification provide, in part, a hydrogelformulation disclosed in the present specification exhibiting a dynamicviscosity. Viscosity is resistance of a fluid to shear or flow caused byeither shear stress or tensile stress. Viscosity describes a fluid'sinternal resistance to flow caused by intermolecular friction exertedwhen layers of fluids attempt to slide by one another and may be thoughtof as a measure of fluid friction. The less viscous the fluid, thegreater its ease of movement (fluidity).

Viscosity can be defined in two ways; dynamic viscosity (μ, although ηis sometimes used) or kinematic viscosity (v). Dynamic viscosity, alsoknown as absolute or complex viscosity, is the tangential force per unitarea required to move one horizontal plane with respect to the other atunit velocity when maintained a unit distance apart by the fluid. The SIphysical unit of dynamic viscosity is the Pascal-second (Pa·s), which isidentical to N·m-2·s. Dynamic viscosity can be expressed as τ=μdvx/dz,where τ=shearing stress, μ=dynamic viscosity, and dvx/dz is the velocitygradient over time. For example, if a fluid with a viscosity of one Pa·sis placed between two plates, and one plate is pushed sideways with ashear stress of one Pascal, it moves a distance equal to the thicknessof the layer between the plates in one second. Dynamic viscositysymbolize by is also used, is measured with various types of rheometers,devices used to measure the way in which a liquid, suspension or slurryflows in response to applied forces.

Kinematic viscosity (v) is the ratio of dynamic viscosity to density, aquantity in which no force is involved and is defined as follows: v=μ/ρ,where μ is the dynamic viscosity ρ is density with the SI unit of kg/m³.Kinematic viscosity is usually measured by a glass capillary viscometeras has an SI unit of m²/s.

The viscosity of a fluid is highly temperature dependent and for eitherdynamic or kinematic viscosity to be meaningful, the referencetemperature must be quoted. For the viscosity values disclosed in thepresent specification, a dynamic viscosity is measured at 1 Pa with acone/plane geometry 2°/40 cm and a temperature of 20° C. Examples of thedynamic viscosity of various fluids at 20° C. is as follows: water isabout 1.0×10⁻³ Pa·s, blood is about 3-4×10⁻³ Pa·s, vegetable oil isabout 60-85×10⁻³ Pa·s, motor oil SE 30 is about 0.2 Pa·s, glycerin isabout 1.4 Pa·s, maple syrup is about 2-3 Pa·s, honey is about 10 Pa·s,chocolate syrup is about 10-25 Pa·s, peanut butter is about 150-250Pa·s, lard is about 1,000 Pa·s, vegetable shortening is about 1,200Pa·s, and tar is about 30,000 Pa·s.

Thus, in an embodiment, a hydrogel formulation comprising a gel phase,the gel phase including hydrogel particles comprising a substantiallysericin-depleted silk fibroin and a carrier phase exhibits a dynamicviscosity. In aspects of this embodiment, a hydrogel compositioncomprising a gel phase, the gel phase including hydrogel particlescomprising a substantially sericin-depleted silk fibroin and a carrierphase exhibits a dynamic viscosity of, e.g., about 10 Pa·s, about 20Pa·s, about 30 Pa·s, about 40 Pa·s, about 50 Pa·s, about 60 Pa·s, about70 Pa·s, about 80 Pa·s, about 90 Pa·s, about 100 Pa·s, about 125 Pa·s,about 150 Pa·s, about 175 Pa·s, about 200 Pa·s, about 225 Pa·s, about250 Pa·s, about 275 Pa·s, about 300 Pa·s, about 400 Pa·s, about 500Pa·s, about 600 Pa·s, about 700 Pa·s, about 750 Pa·s, about 800 Pa·s,about 900 Pa·s, about 1,000 Pa·s, about 1,100 Pa·s, or about 1,200 Pa·s.In other aspects of this embodiment, a hydrogel formulation comprising agel phase, the gel phase including hydrogel particles comprising asubstantially sericin-depleted silk fibroin and a carrier phase exhibitsa dynamic viscosity of, e.g., at most 10 Pa·s, at most 20 Pa·s, at most30 Pa·s, at most 40 Pa·s, at most 50 Pa·s, at most 60 Pa·s, at most 70Pa·s, at most 80 Pa·s, at most 90 Pa·s, at most 100 Pa·s, at most 125Pa·s, at most 150 Pa·s, at most 175 Pa·s, at most 200 Pa·s, at most 225Pa·s, at most 250 Pa·s, at most 275 Pa·s, at most 300 Pa·s, at most 400Pa·s, at most 500 Pa·s, at most 600 Pa·s, at most 700 Pa·s, at most 750Pa·s, at most 800 Pa·s, at most 900 Pa·s, or at most 1000 Pa·s. In yetother aspects of this embodiment, a hydrogel formulation comprising agel phase, the gel phase including hydrogel particles comprising asubstantially sericin-depleted silk fibroin and a carrier phase exhibitsa dynamic viscosity of, e.g., about 10 Pa·s to about 100 Pa·s, about 10Pa·s to about 150 Pa·s, about 10 Pa·s to about 250 Pa·s, about 50 Pa·sto about 100 Pa·s, about 50 Pa·s to about 150 Pa·s, about 50 Pa·s toabout 250 Pa·s, about 100 Pa·s to about 500 Pa·s, about 100 Pa·s toabout 750 Pa·s, about 100 Pa·s to about 1,000 Pas, about 100 Pa·s toabout 1,200 Pa·s, about 300 Pa·s to about 500 Pa·s, about 300 Pa·s toabout 750 Pa·s, about 300 Pa·s to about 1,000 Pa·s, or about 300 Pa·s toabout 1,200 Pa·s.

Aspects of the present specification provide, in part, a hydrogelformulation disclosed in the present specification that is injectable.As used herein, the term “injectable” refers to a hydrogel formulationdisclosed in the present specification having the properties necessaryto administer the composition into a dermal region of an individualusing an injection device with a fine needle. As used herein, the term“fine needle” refers to a needle that is 27 gauge or smaller.Injectability of a hydrogel formulation disclosed in the presentspecification can be accomplished by sizing the hydrogel particles asdiscussed above.

Thus, in an embodiment, a hydrogel formulation comprises a gel phase,the gel phase including hydrogel particles comprising a substantiallysericin-depleted silk fibroin and a carrier phase, wherein theformulation is injectable. In aspect of this embodiment, a hydrogelformulation comprising a gel phase, the gel phase including hydrogelparticles comprising a substantially sericin-depleted silk fibroin and acarrier phase is injectable through a fine needle. In other aspects ofthis embodiment, a hydrogel formulation comprising a gel phase, the gelphase including hydrogel particles comprising a substantiallysericin-depleted silk fibroin and a carrier phase is injectable througha needle of, e.g., about 27 gauge, about 30 gauge, or about 32 gauge. Inyet other aspects of this embodiment, a hydrogel formulation comprisinga gel phase, the gel phase including hydrogel particles comprising asubstantially sericin-depleted silk fibroin and a carrier phase isinjectable through a needle of, e.g., 27 gauge or smaller, 30 gauge orsmaller, or 32 gauge or smaller. In still other aspects of thisembodiment, a hydrogel formulation comprising a gel phase, the gel phaseincluding hydrogel particles comprising a substantially sericin-depletedsilk fibroin and a carrier phase is injectable through a needle of,e.g., about 27 gauge to about 32 gauge.

In aspects of this embodiment, a hydrogel formulation comprises a gelphase, the gel phase including hydrogel particles comprising asubstantially sericin-depleted silk fibroin and a carrier phase can beinjected with an extrusion force of about 60 N, about 55 N, about 50 N,about 45 N, about 40 N, about 35 N, or about 30 N. In other aspects ofthis embodiment, a hydrogel formulation comprising a gel phase, the gelphase including hydrogel particles comprising a substantiallysericin-depleted silk fibroin and a carrier phase can be injected withan extrusion force of about 60 N or less, about 55 N or less, about 50 Nor less, about 45 N or less, about 40 N or less, about 35 N or less,about 30 N or less, about 25 N or less, about 20 N or less, about 15 Nor less, about 10 N or less, or about 5 N or less.

Aspects of the present specification provide, in part, a silk fibroinhydrogel exhibits cohesiveness. Cohesion or cohesive attraction,cohesive force, or compression force is a physical property of amaterial, caused by the intermolecular attraction between like-moleculeswithin the material that acts to unite the molecules. A silk fibroinhydrogel should be sufficiently cohesive as to remain localized to asite of administration. Additionally, in certain applications, asufficient cohesiveness is important for a hydrogel to retain its shape,and thus functionality, in the event of mechanical load cycling. Assuch, in one embodiment, a silk fibroin hydrogel exhibits strongcohesive attraction, on par with water. In another embodiment, a silkfibroin hydrogel exhibits low cohesive attraction. In yet anotherembodiment, a silk fibroin hydrogel exhibits sufficient cohesiveattraction to remain localized to a site of administration. In stillanother embodiment, a silk fibroin hydrogel exhibits sufficient cohesiveattraction to retain its shape. In a further embodiment, a silk fibroinhydrogel exhibits sufficient cohesive attraction to retain its shape andfunctionality.

In aspects of this embodiment, a silk fibroin hydrogel has a compressionforce of about 10 grams-force, about 20 grams-force, about 30grams-force, about 40 grams-force, about 50 grams-force, about 60grams-force, about 70 grams-force, about 80 grams-force, about 90grams-force, about 100 grams-force, about 200 grams-force, about 300grams-force, about 400 grams-force, about 500 grams-force, about 600grams-force, about 700 grams-force, or about 800 grams-force. In otheraspects of this embodiment, a silk fibroin hydrogel has a compressionforce of at least 500 grams-force, at least 600 grams-force, at least700 grams-force, at least 800 grams-force, at least 900 grams-force, atleast 1000 grams-force, at least 1250 grams-force, at least 1500grams-force, at least 1750 grams-force, at least 2000 grams-force, atleast 2250 grams-force, at least 2500 grams-force, at least 2750grams-force, or at least 3000 grams-force. In other aspects of thisembodiment, a silk fibroin hydrogel has a compression force of at most10 grams-force, at most 20 grams-force, at most 30 grams-force, at most40 grams-force, at most 50 grams-force, at most 60 grams-force, at most70 grams-force, at most 80 grams-force, at most 90 grams-force, at most100 grams-force, at most 200 grams-force, at most 300 grams-force, atmost 400 grams-force, at most 500 grams-force, at most 600 grams-force,at most 700 grams-force, or at most 800 grams-force.

In yet other aspects of this embodiment, a silk fibroin hydrogel has acompression force of about 10 grams-force to about 50 grams-force, about25 grams-force to about 75 grams-force, about 50 grams-force to about150 grams-force, about 100 grams-force to about 200 grams-force, about100 grams-force to about 300 grams-force, about 100 grams-force to about400 grams-force, about 100 grams-force to about 500 grams-force, about200 grams-force to about 300 grams-force, about 200 grams-force to about400 grams-force, about 200 grams-force to about 500 grams-force, about200 grams-force to about 600 grams-force, about 200 grams-force to about700 grams-force, about 300 grams-force to about 400 grams-force, about300 grams-force to about 500 grams-force, about 300 grams-force to about600 grams-force, about 300 grams-force to about 700 grams-force, about300 grams-force to about 800 grams-force, about 400 grams-force to about500, about 400 grams-force to about 600, about 400 grams-force to about700, about 400 grams-force to about 800, about 500 grams-force to about600 grams-force, about 500 grams-force to about 700 grams-force, about500 grams-force to about 800 grams-force, about 600 grams-force to about700 grams-force, about 600 grams-force to about 800 grams-force, about700 grams-force to about 800 grams-force, about 1000 grams-force toabout 2000 grams-force, about 1000 grams-force to about 3000grams-force, or about 2000 grams-force to about 3000 grams-force.

Aspects of the present hydrogel formulations provide, in part, asurfactant. As used herein, the term “surfactant” refers to a natural orsynthetic amphiphilic compound. A surfactant can be non-ionic,zwitterionic, or ionic. It is envisioned that any surfactant is usefulin making a hydrogel formulation disclosed in the present specification,with the proviso that a therapeutically effective amount of the hydrogelformulation is recovered using this surfactant amount. Non-limitingexamples of surfactants include polysorbates like polysorbate 20 (TWEEN®20), polysorbate 40 (TWEEN® 40), polysorbate 60 (TWEEN® 60), polysorbate61 (TWEEN® 61), polysorbate 65 (TWEEN® 65), polysorbate 80 (TWEEN® 80),and polysorbate 81 (TWEEN® 81); poloxamers (polyethylene-polypropylenecopolymers), like Poloxamer 124 (PLURONIC® L44), Poloxamer 181(PLURONIC® L61), Poloxamer 182 (PLURONIC® L62), Poloxamer 184 (PLURONIC®L64), Poloxamer 188 (PLURONIC® F68), Poloxamer 237 (PLURONIC® F87),Poloxamer 338 (PLURONIC® L108), Poloxamer 407 (PLURONIC® F127),polyoxyethyleneglycol dodecyl ethers, like BRIJ® 30, and BRIJ® 35;2-dodecoxyethanol (LUBROL®-PX); polyoxyethylene octyl phenyl ether(TRITON® X-100); sodium dodecyl sulfate (SDS);3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS);3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate(CHAPSO); sucrose monolaurate; and sodium cholate. Other non-limitingexamples of surfactant excipients can be found in, e.g., PharmaceuticalDosage Forms and Drug Delivery Systems (Howard C. Ansel et al., eds.,Lippincott Williams & Wilkins Publishers, 7^(th) ed. 1999); Remington:The Science and Practice of Pharmacy (Alfonso R. Gennaro ed.,Lippincott, Williams & Wilkins, 20^(th) ed. 2000); Goodman & Gilman'sThe Pharmacological Basis of Therapeutics (Joel G. Hardman et al., eds.,McGraw-Hill Professional, 10^(th) ed. 2001); and Handbook ofPharmaceutical Excipients (Raymond C. Rowe et al., APhA Publications,4^(th) edition 2003), each of which is hereby incorporated by referencein its entirety.

Thus in an embodiment, a hydrogel formulation comprises a surfactant. Inaspects of this embodiment, a hydrogel formulation comprises apolysorbate, a poloxamer, a polyoxyethyleneglycol dodecyl ether,2-dodecoxyethanol, polyoxyethylene octyl phenyl ether, sodium dodecylsulfate, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate,3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate,sucrose monolaurate; or sodium cholate.

The hydrogel formulations disclosed in the present specification may, ormay not, comprise a viscosity inducing component. A viscosity inducingcomponent is present in an effective amount in increasing the viscosityof the hydrogel formulation. A relatively high viscosity may enhance theability of the present hydrogel formulations maintain the hydrogelparticles in substantially uniform suspension in the compositions forprolonged periods of time, for example, for as long as 1 to 2 years,without requiring resuspension processing. The relatively high viscosityof the present compositions may also have an additional benefit of atleast assisting the compositions to have the ability to have anincreased amount or concentration of the hydrogel particles, whilemaintaining such hydrogel particles in substantially uniform suspensionfor prolonged periods of time.

The presently useful viscosity inducing components preferably are shearthinning components in that as the present hydrogel formulationscontaining such a shear thinning viscosity inducing component is passedor injected through a narrow space, such as 27 gauge needle, under highshear conditions the viscosity of the composition is substantiallyreduced during such passage. After such passage, the composition regainssubstantially its pre-injection viscosity so as to maintain thecorticosteroid component particles in suspension in the eye.

Any suitable viscosity inducing component, for example, may be employedin accordance with the hydrogel formulations disclosed in the presentspecification. The viscosity inducing component is present in an amounteffective in providing the desired viscosity to the hydrogelformulation. Advantageously, the viscosity inducing component is presentin an amount in a range of about 0.5% or about 1.0% to about 5% or about10% or about 20% (w/v) of the hydrogel formulation. The specific amountof the viscosity inducing component employed depends upon a number offactors including, without limitation, the specific viscosity inducingcomponent being employed, the molecular weight of the viscosity inducingcomponent being employed, the viscosity desired for the present hydrogelformulation being produced and/or used and the like factors, such asshear thinning. The viscosity inducing component is chosen to provide atleast one advantage, and preferably multiple advantages, to the presenthydrogel formulation, for example, in terms of each of injectability,viscosity, sustainability of the hydrogel particles in suspension, forexample, in substantially uniform suspension, for a prolonged period oftime without resuspension processing, compatibility with the tissuesinto which the composition is to be placed and the like advantages. Morepreferably, the selected viscosity inducing component is effective toprovide two or more of the above-noted benefits, and still morepreferably to provide all of the above-noted benefits.

The viscosity inducing component preferably comprises a polymericcomponent and/or at least one viscoelastic agent. Examples of usefulviscosity inducing components include, but are not limited to,hyaluronic acid (such as a polymeric hyaluronic acid), carbomers,polyacrylic acid, cellulosic derivatives, polycarbophil,polyvinylpyrrolidone, gelatin, dextrin, polysaccharides, polyacrylamide,polyvinyl alcohol, polyvinyl acetate, derivatives thereof and mixturesand copolymers thereof.

The molecular weight of the presently useful viscosity inducingcomponents may be in a range of about 10,000 Da or less to about2,000,000 Da or more. In one particularly useful embodiment, themolecular weight of the viscosity inducing component is in a range ofabout 100,000 Da or about 200,000 Da to about 1,000,000 Da or about1,500,000 Da. Again, the molecular weight of the viscosity inducingcomponent useful in accordance with the present specification, may varyover a substantial range based on the type of viscosity inducingcomponent employed, and the desired final viscosity of the presentcomposition in question, as well as, possibly one or more other factors.

In one embodiment, a viscosity inducing component is a polymerichyaluronate component, for example, a metal hyaluronate component,preferably selected from alkali metal hyaluronates, alkaline earth metalhyaluronates and mixtures thereof, and still more preferably selectedfrom sodium hyaluronates, and mixtures thereof. The molecular weight ofsuch hyaluronate component (i.e. a polymeric hyaluronic acid) preferablyis in a range of about 50,000 Da or about 100,000 Da to about 1,300,000Da or about 2,000,000 Da. In one embodiment, the present compositionsinclude a polymeric hyaluronate component in an amount in a range about0.05% to about 0.5% (w/v). In a further useful embodiment, thehyaluronate component is present in an amount in a range of about 1% toabout 4% (w/v) of the composition. In this latter case, the very highpolymer viscosity forms a gel that slows particle sedimentation rate tothe extent that often no resuspension processing is necessary over theestimated shelf life, for example, at least about 2 years, of thecomposition. Such a formulation may be marketed in pre-filled syringessince the gel cannot be easily removed by a needle and syringe from abulk container. Pre-filled syringes have the advantages of conveniencefor the injector and the safety which results from less handling.

Aspects of the present specification provide, in part, a hydrogelformulation disclosed in the present specification that is apharmaceutical hydrogel formulation. As used herein, the term“pharmaceutical hydrogel formulation” is synonymous with“pharmaceutically-acceptable hydrogel formulation” and refers to atherapeutically effective concentration of hydrogel formulation, suchas, e.g., any of the hydrogel particles or pharmaceutically-acceptabledrugs disclosed in the present specification. A pharmaceutical hydrogelformulation is useful for medical and veterinary applications. Apharmaceutical hydrogel formulation may be administered to a individualalone, or in combination with other supplementary active ingredients,agents, drugs or hormones.

Aspects of the present specification provide, in part, a hydrogelformulation disclosed in the present specification that is apharmaceutical hydrogel formulation comprising a pharmacologicallyacceptable excipient. As used herein, the term “pharmacologicallyacceptable excipient” is synonymous with “pharmacological excipient” or“excipient” and refers to any excipient that has substantially no longterm or permanent detrimental effect when administered to mammal andencompasses compounds such as, e.g., stabilizing agent, a bulking agent,a cryo-protectant, a lyo-protectant, an additive, a vehicle, a carrier,a diluent, or an auxiliary. An excipient generally is mixed with anactive ingredient, or permitted to dilute or enclose the activeingredient and can be a solid, semi-solid, or liquid agent. It is alsoenvisioned that a pharmaceutical hydrogel formulation can include one ormore pharmaceutically acceptable excipients that facilitate processingof a hydrogel formulation into pharmaceutically acceptable hydrogelformulation. Insofar as any pharmacologically acceptable excipient isnot incompatible with a hydrogel formulation, its use inpharmaceutically acceptable compositions is contemplated. Non-limitingexamples of pharmacologically acceptable excipients can be found in,e.g., Pharmaceutical Dosage Forms and Drug Delivery Systems (Howard C.Ansel et al., eds., Lippincott Williams & Wilkins Publishers, 7^(th) ed.1999); Remington: The Science and Practice of Pharmacy (Alfonso R.Gennaro ed., Lippincott, Williams & Wilkins, 20^(th) ed. 2000); Goodman& Gilman's The Pharmacological Basis of Therapeutics (Joel G. Hardman etal., eds., McGraw-Hill Professional, 10^(th) ed. 2001); and Handbook ofPharmaceutical Excipients (Raymond C. Rowe et al., APhA Publications,4^(th) edition 2003), each of which is hereby incorporated by referencein its entirety.

It is further envisioned that a pharmaceutical hydrogel formulationdisclosed in the present specification may optionally include or notinclude, without limitation, other pharmaceutically acceptablecomponents (or pharmaceutical components), including, withoutlimitation, buffers, preservatives, tonicity adjusters, salts,antioxidants, osmolality adjusting agents, emulsifying agents, wettingagents, sweetening or flavoring agents, and the like.

Pharmaceutically acceptable buffer is any buffer that can be used toprepare a pharmaceutical hydrogel formulation disclosed in the presentspecification, provided that the resulting preparation ispharmaceutically acceptable. Non-limiting examples of pharmaceuticallyacceptable buffers include acetate buffers, borate buffers, citratebuffers, neutral buffered salines, phosphate buffers, and phosphatebuffered salines. Any concentration of a pharmaceutically acceptablebuffer can be useful in formulating a pharmaceutical compositiondisclosed in the present specification, with the proviso that atherapeutically effective amount of the matrix polymer active ingredientis recovered using this effective concentration of buffer. Non-limitingexamples of concentrations of physiologically-acceptable buffers occurwithin the range of about 0.1 mM to about 900 mM. The pH ofpharmaceutically acceptable buffers may be adjusted, provided that theresulting preparation is pharmaceutically acceptable. It is understoodthat acids or bases can be used to adjust the pH of a pharmaceuticalcomposition as needed. Any buffered pH level can be useful informulating a pharmaceutical hydrogel formulation, with the proviso thata therapeutically effective amount of the hydrogel formulation isrecovered using this effective pH level. Non-limiting examples ofphysiologically-acceptable pH occur within the range of about pH 5.5 toabout pH 8.5.

Pharmaceutically acceptable antioxidants include, without limitation,sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylatedhydroxyanisole and butylated hydroxytoluene. Pharmaceutically acceptablepreservatives include, without limitation, benzalkonium chloride,chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuricnitrate, a stabilized oxy chloro composition, such as, e.g., PURITE® andchelants, such as, e.g., DTPA or DTPA-bisamide, calcium DTPA, andCaNa-DTPA-bisamide.

Tonicity adjustors useful in a pharmaceutical hydrogel formulationinclude, without limitation, salts such as, e.g., sodium chloride andpotassium chloride; and glycerin. The pharmaceutical hydrogelformulation may be provided as a salt and can be formed with many acids,including but not limited to, hydrochloric, sulfuric, acetic, lactic,tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueousor other protonic solvents than are the corresponding free base forms.It is understood that these and other substances known in the art ofpharmacology can be included in a pharmaceutical hydrogel formulationdisclosed in the present specification. Other non-limiting examples ofpharmacologically acceptable components can be found in, e.g., Ansel,supra, (1999); Gennaro, supra, (2000); Hardman, supra, (2001); and Rowe,supra, (2003), each of which is hereby incorporated by reference in itsentirety.

A hydrogel formulation disclosed in the present specification generallyis administered as a pharmaceutical acceptable hydrogel formulation. Asused herein, the term “pharmaceutically acceptable” means any molecularentity or composition that does not produce an adverse, allergic orother untoward or unwanted reaction when administered to an individual.

The silk hydrogels as provided for herein may be filled into syringesand sterilized. For example, the pulverized gel saline formulation maybe loaded into 1 mL syringes and capped with 26 g needles. These may bestored, e.g., at 4° C., and sterilized, for example, in pouches byexposure to gamma radiation for a dose of 25 kGy. After sterilization,the syringes may be stored until later use under a temperature rangefrom 4° C. to 37° C. until use.

Thus, for example, a formulation may be processed by obtaining an 8%(w/v) silk solution, adding ethanol/23RGD to generate a firm 4%-6% (w/v)silk fibroin hydrogel, allowing this to stand for at least 24 hours,rinsing the gel in water to remove residual free enhancer (both 23RGDand ethanol), adding saline to the gel and homogenizing the suspension,tailoring the suspension viscosity with gel concentration, particlesize, and saline content, milling the gel to a desired particle sizethat makes the gel injectable through a 30 g needle, loading the gelinto a syringe, and sterilizing the gel with gamma irradiation.

The silk fibroin hydrogels disclosed in the present specification can beused in a variety of medical uses, including, without limitation fillersfor tissue space, templates for tissue reconstruction or regeneration,scaffolds for cells in tissue engineering applications and for diseasemodels, a surface coating to improve medical device function, or as aplatform for drug delivery.

In any of the uses described below, silk fibroin gels could be combinedwith cells for purposes of a biologically enhanced repair. Cells couldbe collected from a multitude of hosts including but not limited tohuman autograft tissues, transgenic mammals, or bacterial cultures(possibly for use as a probiotic treatment). More specifically, humancells used could consist of stem cells, osteocytes, fibroblasts,neuronal cells, lipocytes, and assorted immunocytes. These cells wouldbe best added after rinsing of the silk gel material itself. They couldbe seeded upon the surface of a solid implant material or in the case ofcomminuted gel injectables, blended into the silk gel particles, carriersolution, or mixture of silk gel particles and carrier solution prior toinjection or implantation.

In addition, therapeutic agents, pharmaceuticals, or specific growthfactors added to the gel for purposes of improved outcome could beintroduced at any or a combination of several points throughout the silkgel production process. These factors could be added to silk solution orthe accelerant phase prior to gelation induction, they could be soakedinto the gel during the accelerant rinsing process, or they could becoated onto the bulk gel following rinsing. Gels which are milled andmake use of a carrier fluid could also have an agent soaked into the gelfollowing milling, coated onto the gel following milling, or introducedinto the carrier fluid before or after blending with the gel material.

The silk fibroin hydrogels disclosed in the present specification can beused as tissue space fillers, such as, e.g., a dermal filler. Oneembodiment the invention provides a dermal filler to provide dermalbulking to improve skin appearance or condition, including, withoutlimitation, rehydrating the skin, providing increased elasticity to theskin, reducing skin roughness, making the skin tauter, reducing oreliminating stretch lines or marks, giving the skin better tone, shine,brightness and/or radiance to reduce paleness, reducing or eliminatingwrinkles in the skin, providing wrinkle resistance to the skin, and thelike. A dermal filler comprising a silk fibroin hydrogel can bemodulation for gel hardness and opacity through alteration of silkconcentration and formulatory method. Most likely formulatory strategywould entail casting of a bulk silk gel, about 2% (w/v) to about 6%(w/v) in silk fibroin concentration containing a RGD component in therange of about 0.1 to about 10 moles of RGD per mole of silk in the gelmaterial. This gel would in turn be milled in such a manner as to beinjectable through a 26-30 g needle. This milled gel should then beblended with a carrier fluid, saline for example, in order to allow foran appropriate extrusion force of less than 40 N (nominal deliverableinjection force for a human hand). Likely carrier content in the case ofsaline should be on the order of 5% to 50% (v/v). For example, higheramount (>25%) of saline addition may be required for applications to insuperficial dermal regions, such as, e.g., reconstructive or cosmeticapplications to the facial region of an individual. Additional benefitwould be derived from infusion of this carrier fluid or gel with ananalgesic or other therapeutic compound. In addition, the silk fibroingel could be combined with cells for purposes of a biologically enhancedrepair.

Aspects of the present specification provide, in part, a method ofimproving a condition of skin in an individual in need thereof, themethod comprising the steps of administering a silk fibroin hydrogelformulation disclosed in the present specification into a dermal regionof the individual, wherein the formulation comprises a gel phase, thegel phase including hydrogel particles comprising a substantiallysericin-depleted silk fibroin and an amphiphilic peptide; and a carrierphase; and wherein the administration improves the condition.

Aspects of the present specification provide, in part, a method ofimproving an appearance of skin in an individual in need thereof, themethod comprising the steps of administering a silk fibroin hydrogelformulation disclosed in the present specification into a dermal regionof the individual, wherein the formulation comprises a gel phase, thegel phase including hydrogel particles comprising a substantiallysericin-depleted silk fibroin and an amphiphilic peptide; and a carrierphase; and wherein the administration improves the appearance of theskin.

Aspects of the present invention provide, in part, an appearance ofskin. Non-limiting examples of a skin appearance include skin wrinkles,lack of skin tautness, skin stretch line and/or marks, and the like.

Aspects of the present invention provide, in part, improving a skinappearance. Non-limiting examples of improving a skin appearance includereducing or eliminating wrinkles in the skin, making the skin tauter,reducing or eliminating stretch lines or marks, giving the skin bettertone, shine, brightness and/or radiance to reduce paleness, and thelike.

Aspects of the present invention provide, in part, a condition of skin.Non-limiting examples of a skin condition include dehydration, lack ofskin elasticity, roughness, lack of skin tautness, skin stretch lineand/or marks, skin paleness, skin wrinkles, and the like.

Aspects of the present invention provide, in part, improving a skincondition. Non-limiting examples of improving a skin condition includerehydrating the skin, providing increased elasticity to the skin,reducing skin roughness, making the skin tauter, reducing or eliminatingstretch lines or marks, giving the skin better tone, shine, brightnessand/or radiance to reduce paleness, reducing or eliminating wrinkles inthe skin, providing wrinkle resistance to the skin, and the like.

Thus, in an embodiment, a method of treating a skin condition comprisesthe step of administering to an individual suffering from a skincondition a silk fibroin hydrogel formulation wherein the formulationcomprises a gel phase, the gel phase including hydrogel particlescomprising a substantially sericin-depleted silk fibroin and anamphiphilic peptide; and a carrier phase, and wherein the administrationof the composition improves the skin condition, thereby treating theskin condition. In an aspect of this embodiment, a method of treatingskin dehydration comprises the step of administering to an individualsuffering from skin dehydration a silk fibroin hydrogel formulationwherein the formulation comprises a gel phase, the gel phase includinghydrogel particles comprising a substantially sericin-depleted silkfibroin and an amphiphilic peptide; and a carrier phase, and wherein theadministration of the composition rehydrates the skin, thereby treatingskin dehydration. In another aspect of this embodiment, a method oftreating a lack of skin elasticity comprises the step of administeringto an individual suffering from a lack of skin elasticity a silk fibroinhydrogel formulation wherein the formulation comprises a gel phase, thegel phase including hydrogel particles comprising a substantiallysericin-depleted silk fibroin and an amphiphilic peptide; and a carrierphase, and wherein the administration of the composition increases theelasticity of the skin, thereby treating a lack of skin elasticity. Inyet another aspect of this embodiment, a method of treating skinroughness comprises the step of administering to an individual sufferingfrom skin roughness a silk fibroin hydrogel formulation wherein theformulation comprises a gel phase, the gel phase including hydrogelparticles comprising a substantially sericin-depleted silk fibroin andan amphiphilic peptide; and a carrier phase, and wherein theadministration of the composition decreases skin roughness, therebytreating skin roughness. In still another aspect of this embodiment, amethod of treating a lack of skin tautness comprises the step ofadministering to an individual suffering from a lack of skin tautness asilk fibroin hydrogel formulation wherein the formulation comprises agel phase, the gel phase including hydrogel particles comprising asubstantially sericin-depleted silk fibroin and an amphiphilic peptide;and a carrier phase, and wherein the administration of the compositionmakes the skin tauter, thereby treating a lack of skin tautness.

In a further aspect of this embodiment, a method of treating a skinstretch line or mark comprises the step of administering to anindividual suffering from a skin stretch line or mark a silk fibroinhydrogel formulation wherein the formulation comprises a gel phase, thegel phase including hydrogel particles comprising a substantiallysericin-depleted silk fibroin and an amphiphilic peptide; and a carrierphase, and wherein the administration of the composition reduces oreliminates the skin stretch line or mark, thereby treating a skinstretch line or mark. In another aspect of this embodiment, a method oftreating skin paleness comprises the step of administering to anindividual suffering from skin paleness a silk fibroin hydrogelformulation wherein the formulation comprises a gel phase, the gel phaseincluding hydrogel particles comprising a substantially sericin-depletedsilk fibroin and an amphiphilic peptide; and a carrier phase, andwherein the administration of the composition increases skin tone orradiance, thereby treating skin paleness. In another aspect of thisembodiment, a method of treating skin wrinkles comprises the step ofadministering to an individual suffering from skin wrinkles a silkfibroin hydrogel formulation wherein the formulation comprises a gelphase, the gel phase including hydrogel particles comprising asubstantially sericin-depleted silk fibroin and an amphiphilic peptide;and a carrier phase, and wherein the administration of the compositionreduces or eliminates skin wrinkles, thereby treating skin wrinkles. Inyet another aspect of this embodiment, a method of treating skinwrinkles comprises the step of administering to an individual a silkfibroin hydrogel formulation wherein the formulation comprises a gelphase, the gel phase including hydrogel particles comprising asubstantially sericin-depleted silk fibroin and an amphiphilic peptide;and a carrier phase, and wherein the administration of the compositionmakes the skin resistant to skin wrinkles, thereby treating skinwrinkles.

Aspects of the present invention provide, in part, a dermal region. Asused herein, the term “dermal region” refers to the region of skincomprising the epidermal-dermal junction and the dermis including thesuperficial dermis (papillary region) and the deep dermis (reticularregion). The skin is composed of three primary layers: the epidermis,which provides waterproofing and serves as a barrier to infection; thedermis, which serves as a location for the appendages of skin; and thehypodermis (subcutaneous adipose layer). The epidermis contains no bloodvessels, and is nourished by diffusion from the dermis. The main type ofcells which make up the epidermis are keratinocytes, melanocytes,Langerhans cells and Merkels cells.

The dermis is the layer of skin beneath the epidermis that consists ofconnective tissue and cushions the body from stress and strain. Thedermis is tightly connected to the epidermis by a basement membrane. Italso harbors many Mechanoreceptor/nerve endings that provide the senseof touch and heat. It contains the hair follicles, sweat glands,sebaceous glands, apocrine glands, lymphatic vessels and blood vessels.The blood vessels in the dermis provide nourishment and waste removalfrom its own cells as well as from the Stratum basale of the epidermis.The dermis is structurally divided into two areas: a superficial areaadjacent to the epidermis, called the papillary region, and a deepthicker area known as the reticular region.

The papillary region is composed of loose areolar connective tissue. Itis named for its fingerlike projections called papillae that extendtoward the epidermis. The papillae provide the dermis with a “bumpy”surface that interdigitates with the epidermis, strengthening theconnection between the two layers of skin. The reticular region liesdeep in the papillary region and is usually much thicker. It is composedof dense irregular connective tissue, and receives its name from thedense concentration of collagenous, elastic, and reticular fibers thatweave throughout it. These protein fibers give the dermis its propertiesof strength, extensibility, and elasticity. Also located within thereticular region are the roots of the hair, sebaceous glands, sweatglands, receptors, nails, and blood vessels. Tattoo ink is held in thedermis. Stretch marks from pregnancy are also located in the dermis.

The hypodermis is not part of the skin, and lies below the dermis. Itspurpose is to attach the skin to underlying bone and muscle as well assupplying it with blood vessels and nerves. It consists of looseconnective tissue and elastin. The main cell types are fibroblasts,macrophages and adipocytes (the hypodermis contains 50% of body fat).Fat serves as padding and insulation for the body.

Aspects of the present invention provide, in part, an individual. Asused herein, the term “individual” refers to any mammal including ahuman being.

Aspects of the present invention provide, in part, administering a silkfibroin hydrogel formulation disclosed in the present specification. Asused herein, the term “administering” means any delivery mechanism thatprovides a silk fibroin hydrogel formulation wherein the formulationcomprises a gel phase, the gel phase including hydrogel particlescomprising a substantially sericin-depleted silk fibroin and anamphiphilic peptide; and a carrier phase, to an individual thatpotentially results in a clinically, therapeutically, or experimentallybeneficial result. The actual delivery mechanism used to administer asilk fibroin hydrogel formulation disclosed in the present specificationto an individual can be determined by a person of ordinary skill in theart by taking into account factors, including, without limitation, thetype of skin condition, the location of the skin condition, the cause ofthe skin condition, the severity of the skin condition, the degree ofrelief desired, the duration of relief desired, the particular silkfibroin hydrogel formulation used, the rate of excretion of theparticular silk fibroin hydrogel formulation used, the pharmacodynamicsof the particular silk fibroin hydrogel formulation used, the nature ofthe other compounds included in the particular silk fibroin hydrogelformulation used, the particular route of administration, the particularcharacteristics, history and risk factors of the individual, such as,e.g., age, weight, general health and the like, or any combinationthereof.

Thus, in an embodiment, a silk fibroin hydrogel formulation wherein theformulation comprises a gel phase, the gel phase including hydrogelparticles comprising a substantially sericin-depleted silk fibroin andan amphiphilic peptide; and a carrier phase, is administered to a skinregion of an individual. In an aspect of this embodiment, a silk fibroinhydrogel formulation wherein the formulation comprises a gel phase, thegel phase including hydrogel particles comprising a substantiallysericin-depleted silk fibroin and an amphiphilic peptide; and a carrierphase, is administered to a skin region of an individual by injection.In another aspect of this embodiment, a silk fibroin hydrogelformulation wherein the formulation comprises a gel phase, the gel phaseincluding hydrogel particles comprising a substantially sericin-depletedsilk fibroin and an amphiphilic peptide; and a carrier phase, isadministered to a skin region of an individual by injection into adermal region. In aspects of this embodiment, a silk fibroin hydrogelformulation wherein the formulation comprises a gel phase, the gel phaseincluding hydrogel particles comprising a substantially sericin-depletedsilk fibroin and an amphiphilic peptide; and a carrier phase, isadministered to a skin region of an individual by injection into, e.g.,an epidermal-dermal junction region, a papillary region, a reticularregion, or any combination thereof.

In another embodiment, the invention provides a dermal filler to providedermal bulking to reconstruct or augment a soft tissue body part, suchas, e.g., a lip, a breast, a breast part like the nipple, a muscle, orany other soft body part where adipose and/or connective tissue is usedto provide shape, insulation, or other biological function. In fillersused for these applications, the silk fibroin concentration and theamount of saline addition to silk fibroin hydrogel may be adjusted tofit the relevant constraints of a given biological environment. Forexample, a gel for breast augmentation would call for modulation of gelhardness and longevity through alteration of silk concentration andformulatory method. Most likely formulatory strategy would entailcasting of a bulk silk gel, about 4% (w/v) to about 8% (w/v) in silkfibroin concentration containing a RGD component in the range of about0.1 to about 10 moles of RGD per mole of silk in the gel material.Likely carrier content in the case of saline should be on the order of0% to 25% (v/v). Such formulatory strategies should also consider otherfactors such as, e.g., defect type, defect size and needs for a specificdepth of administration of the filler. The particles size and uniformityof the hydrogels can be controlled so that the tissue implantation canbe accomplished through injection. For example, for dermal injection andlip augmentation, a syringe needle sized 26 g-30 g would be used. Inapplications involving large quantities of filler, e.g., breastreconstruction or augmentation, a larger particle size and a larger boreneedle like 23 g-27 g may be used to administer the filer. This milledgel should then be blended with a carrier fluid, saline for example, inorder to allow for an appropriate extrusion force of less than 40 N(nominal deliverable injection force for a human hand). Additionalbenefit would be derived from infusion of this carrier fluid or gel withan analgesic or other therapeutic compound. In addition, a silk fibroinhydrogel formulation disclosed in the present specification can be usedto fill an expandable implantable medical device, such as, e.g., anexpandable breast implant shell.

Aspects of the present specification provide, in part, a method of softtissue reconstruction or augmentation, the method comprising the step ofadministering a silk fibroin hydrogel formulation to a soft tissueregion of an individual in need of soft tissue reconstruction oraugmentation; wherein the formulation comprises a gel phase, the gelphase including hydrogel particles comprising a substantiallysericin-depleted silk fibroin and an amphiphilic peptide; and a carrierphase.

Aspects of the present specification provide, in part, a method of softtissue reconstruction or augmentation, the method comprising the step ofplacing an implantable medical device into a soft tissue region of anindividual at the desired location; and expanding the device by puttinga silk fibroin hydrogel formulation into the device, wherein theformulation comprises a gel phase, the gel phase including hydrogelparticles comprising a substantially sericin-depleted silk fibroin andan amphiphilic peptide; and a carrier phase, wherein filling the medicaldevice reconstructs or augments the soft tissue.

Aspects of the present specification provide, in part, a soft tissue.Non-limiting examples of the tissue include a lip, a breast, a breastpart like the nipple, a muscle, or any other soft body part whereadipose and/or connective tissue is used to provide shape, insulation,or other biological function.

Soft tissue reconstruction is the rebuilding of a soft tissue structurethat was severely damaged or lost, e.g., by a dramatic accident orsurgical removal. For example, breast reconstruction is the rebuildingof a breast, usually in women. It involves using autologous tissue orprosthetic material to construct a natural-looking breast. Often thisincludes the reformation of a natural-looking areola and nipple. Thisprocedure involves the use of implants or relocated flaps of thepatient's own tissue.

Soft tissue augmentation is the altering of a soft tissue structureusually to improve the cosmetic or aesthetic appearance of the softtissue. For example, breast augmentation (also known as breastaugmentation, breast enlargement, mammoplasty enlargement, augmentationmammoplasty) alters the size and shape of a woman's breasts to improvethe cosmetic or aesthetic appearance of the woman.

Subdermal administration refers to administering the hydrogelformulations disclosed in the present specification below the dermallayer, e.g., in the hypodermis or in muscle tissue.

The silk fibroin hydrogels disclosed in the present specification can beused in conjunction with interventional radiology embolizationprocedures for blocking abnormal blood (artery) vessels (e.g., for thepurpose of stopping bleeding) or organs (to stop the extra function e.g.embolization of the spleen for hypersplenism) including uterine arteryembolization for percutaneous treatment of uterine fibroids. Modulationof gel hardness and longevity would be done through alteration of silkconcentration and formulatory method. Most likely formulatory strategywould entail casting of a bulk silk gel, about 1% (w/v) to about 4%(w/v) in silk concentration containing a RGD component in the range ofabout 0.01 to about 3 moles of RGD per mole of silk fibroin in the gelmaterial. This gel would in turn be milled in such a manner as to beinjectable through a 26-30 g needle. This milled gel might then beblended with a carrier fluid, saline for example, in order to allow foran appropriate extrusion force of less than 40 N (nominal deliverableinjection force for a human hand). Carrier content in the case of salineshould be on the order of 0% to about 25% (v/v). Additional benefitwould be derived from infusion of this carrier fluid or gel with atherapeutic compound such as a clotting agent. In addition, the silkfibroin gel could be combined with cells for purposes of a biologicallyenhanced repair.

The silk fibroin hydrogels disclosed in the present specification can beused to repair void space created by spine disk nucleus removal surgeryto help maintain the normal distance between the adjacent vertebralbodies. As a further alternative, silk hydrogels with a higherconcentration of silk protein (e.g., 20% (w/v)) can be generated in acustom designed mold. The resulting device can be implanted withoutcarrier fluid or milling as a replacement of diseased vertebral diskremoved by surgical procedure. For this application, the silk hydrogelcan be further chemically or physically cross-linked to gain strongermechanical properties. In addition, the silk fibroin gel could becombined with cells for purposes of a biologically enhanced repair.

In an aspect of this embodiment it would be desirable to employ avertebral disc filler comprising a silk concentration of about 4% toabout 6% (w/v) silk gel. This material would be either milled to aninjectable after casting and blended with 0% to about 20% (v/v) carrierfluid and injected into a ruptured disc during gelation. Appropriate RGDcontent for this material would be on the order of about 0.0001 to about0.01 moles of RGD per mole of silk in the gel material or on the orderof about 5 to about 100 moles of RGD per mole of silk in the gel. Asmall RGD component or very large RGD component could both be veryuseful in discouraging cell proliferation either through denial ofbinding motifs or through provision of so many as to anchor cells anddeny the ability to move and proliferate.

In another aspect of this embodiment it would be desirable to cast asilk gel in an appropriately shaped annular mold for replacement of thedisk entirely. This gel would employ a silk concentration of about 6%(w/v) to about 10% (w/v) within the gel material itself. An appropriateRGD content would be on the order of about 0.1 to about 3 moles of RGDper mole of silk to encourage host tissue ingrowth into the material.This device could then be implanted in situ.

In another aspect of this embodiment, it would be desirable to employ asilk gel which is injected into the defective disc and gels in place.This gel would employ a silk concentration of about 4% (w/v) to about10% (w/v) within the gel material. Accelerant could be mixed with silksolution before, during, or after injection into the site of interest. Alow concentration of accelerant would be desirable for this application.Appropriate RGD content for this material would be on the order of about0.0001 to about 0.01 moles of RGD per mole of silk in the gel materialor on the order of about 5 to about 100 moles of RGD per mole of silk inthe gel. A small RGD component or very large RGD component could both bevery useful in discouraging cell proliferation either through denial ofbinding motifs or through provision of so many as to anchor cells anddeny the ability to move and proliferate.

The silk fibroin hydrogels disclosed in the present specification can beused to fill up the vitreous cavity to support the eyeball structure andmaintain the retina's position. For this application, the implant wouldcomprise low silk fibroin concentration, be highly transparency, andcould be modified to be more bio-inert, i.e., inhibit cell ingrowth andproliferation, through further treatment, for example, through high23RGD dosing. For example, silk fibroin gel used as a filler for thevitreous cavity would entail casting of a bulk silk gel of about 0.5%(w/v) to about 2% (w/v) in silk concentration containing a RGD componentin the range of 0.0001 to 1 moles of RGD per mole of silk in the gelmaterial or on the order of 5 to 100 moles of RGD per mole of silk inthe gel. A small RGD component or very large RGD component could both bevery useful in discouraging cell proliferation either through denial ofbinding motifs or through provision of so many as to anchor cells anddeny the ability to move and proliferate. This gel might be milled tobecome injectable or could be injectable as a bulk depending up onmaterial hardness, though it should be injectable through a 26-30 gneedle with an appropriate extrusion force of less than 40 N. Likelycarrier content should be on the order of 0% to about 95% (v/v). Carriercomposition could be any biologically or anatomically useful liquid andmay consist of saline, hyaluronic acid gel, and host or non-host derivedbiological fluids.

The silk fibroin hydrogels disclosed in the present specification can beused as templates for tissue reconstruction or regeneration. In general,high concentration silk fibroin hydrogels with strong mechanicalproperties and elasticity may be generated in a custom designed moldchamber to form in to the desired shape required. In a case where silkgel is used as a template for tissue reconstruction or regeneration,ultimate application of the material should be considered in selecting aformulation. The device can cast and molded or cast and reshaped, thenimplanted for the tissue reconstruction. In addition, the silk fibroingel could be combined with cells for purposes of a biologically enhancedrepair.

In one embodiment the silk hydrogel can be implanted to the location ofcartilage (like menisci or meniscal cartilage) or bone defect. The silkhydrogel device would facilitate cartilage/bone cell ingrowth andproliferation and support collagen matrix deposition thus to improvecartilage/bone repair. In another aspect, prior to implantation donorcartilage cells can be seeded or mixed with silk hydrogel and culturedin vitro to expand cell population and to develop cartilage tissue thusto short the healing time period. For this application, specific growthfactors such as TGF-β or bone morphogenic proteins (BMPs) which supportcartilage or bone tissue formation, respectively, may be added into silkhydrogel. Silk gels used for replacement of bone or cartilage defectshould be cast as a silk concentration of about 6% (w/v) to about 10%(w/v) silk fibroin. The RGD component should be in a range from about0.01 to about 3 moles of RGD per mole of silk in the gel material. Thesematerials could be cast in a mold and thereby shaped to an appropriateapplication or could be cast in a generic shape and further refined bythe end-user of the material prior to implantation.

In another embodiment, the silk fibroin hydrogels can be used for facialplastic surgery, such as, e.g., nose reconstruction. The formulatorystrategy discussed above for repairing a cartilage/bone defect wouldalso be applicable for this application.

The silk fibroin hydrogels disclosed in the present specification can beused as scaffolds to support cell growth for tissue engineering ordisease model research applications.

In one aspect, the hydrogel scaffolds comprising cells can be use inmethods of promoting wound healing or wound closure, for example, at anincision site. The methods generally comprise implanting a hydrogel, forexample a bioerodible or bioresorbable hydrogel as disclosed in thepresent specification, at the wound or incision site and allowing thewound or incision to heal while the implant is eroded or absorbed in thebody and is replaced with the individual's own viable tissue. Themethods may further comprise the step of seeding the hydrogel withviable cellular material, either from the individual or from a donor,prior to or during implantation.

In another aspect, the hydrogel scaffolds comprising cells can be use inmethods of augmenting or reconstructing the breast of a human being. Forexample, a method for enhancing support of a conventional breastimplant, such as, by enhancing support of the lower pole position of abreast implant. As another example, the method generally comprises thesteps of implanting a hydrogel scaffold near or in proximity to a breastimplant, for example, a conventional breast implant, and seeding thehydrogel with viable cellular material prior to or during implantation.As yet another example, a hydrogel scaffold is used to partially orcompletely cover a breast implant to provide a beneficial interface withhost tissue and to reduce the potential for malpositioning or capsularcontracture.

In yet another aspect, the hydrogel scaffolds comprising cells can beuse in methods of providing a beneficial interface between host tissueand a prostheses, for example, a breast implant. In some embodiments,the matrices are structured to be effective to reduce the potential formalpositioning or capsular contracture of breast implants. For example,methods are provided for augmenting or reconstructing a human breast,the methods generally comprising: providing a partial or completecovering of breast implants wherein the partial or complete coveringcomprises a hydrogel scaffold comprising cells. In some embodiments, thehydrogel is a wrap-like configuration on a conventional silicone orsaline filled conventional breast implant. The methods may furthercomprise the step of seeding the hydrogel with viable cellular materialprior to or during implantation.

In still another aspect, the silk fibroin hydrogel scaffolds disclosedin the present specification can be used as the scaffold for cellsuseful for peripheral nerve repair. Silk hydrogels can be implanted tothe location of the nerve defect with or without additional device toaid the connection to the nerve ends. For this approach, specific growthfactors such as nerve growth factor (NGF), which supports nerveregeneration may be added. Alternatively, nerve cells may be mixed intosilk hydrogel and culture expanded in vitro before implantation. Thereare two possible formulatory strategies in a case where silk fibroin gelis used as a template for peripheral nerve repair. The first involvesuse of a softer silk gel of about 0.5 (w/v) to about 3% (w/v) silkeither shaped cast as a length of nerve scaffold or else milled to beextrudable as a length of nerve scaffold. This material would be infusedwith appropriate therapeutic factors according to the methods describedabove. The second likely formulation of this template would be use ofthe above described soft gel encased in a sheath or conduit of higherstrength silk fibroin gel of about 3 to about 8% silk. This might begenerated through a co-casting technique or by filling a cast conduitwith an injectable core gel material. In the case of all gels an RGDcontent of about 0.01 to about 3 moles of RGD per mole of silk would beappropriate. If a carrier fluid were employed, a concentration between0% and about 25% (v/v) would be most appropriate.

The cells can be seeded upon the surface of a solid implant materialcomprising a hydrogel disclosed in the present specification using avariety of methods. For example, a hydrogel scaffold can be submersed inan appropriate growth medium for the cells of interest, and thendirectly exposed to the cells. The cells are allowed to proliferate onthe surface and interstices of the hydrogel. The hydrogel is thenremoved from the growth medium, washed if necessary, and implanted.Alternatively, the cells can be placed in a suitable buffer or liquidgrowth medium and drawn through a hydrogel scaffold by using vacuumfiltration. Cells can also be admixed with a precursor of a hydrogelscaffold, and the hydrogel scaffold can then be constructed around thecells, capturing at least some of the cells within the hydrogel scaffoldnetwork. In another embodiment, the cells of interest are dissolved intoan appropriate solution (e.g., a growth medium or buffer) and thensprayed onto a hydrogel scaffold while the hydrogel scaffold is beingformed by electrospinning. This method is particularly suitable when ahighly cellularized matrix is desired. Cells can also be electrosprayedonto a hydrogel scaffold during electrospinning. As presently described,electrospraying involves subjecting a cell-containing solution with anappropriate viscosity and concentration to an electric field sufficientto produce a spray of small charged droplets of solution that containcells.

In certain cases, a solid hydrogel scaffold can be made as a porousmaterial comprising a hydrogel defined by an interconnected array ofpores. The size of the pores comprising an interconnected array of poresshould be of a size sufficient to facilitate tissue ingrowth. Suchporous hydrogel matrix can be made using standard procedures such as,porogens or other leachable materials. Such materials include, withoutlimitation, salts like sodium chloride, potassium chloride, calciumchloride, sodium tartrate, sodium citrate, and the like; biocompatiblemono and disaccharides like glucose, fructose, dextrose, maltose,lactose and sucrose); polysaccharides like starch, alginate, chitosan;and water soluble proteins like gelatin and agarose. Porogens and otherleachable materials can be removed by immersing the hydrogel with theleachable material in a solvent in which the particle is soluble for asufficient amount of time to allow leaching of substantially all of theparticles, but which does not dissolve or detrimentally alter thehydrogel. In one embodiment, the hydrogel can be dried after theleaching process is complete at a low temperature and/or vacuum tominimize hydrolysis of the matrix unless accelerated degradation of thematrix is desired. Methods useful for making porogens or other leachablematerials as well as methods of making a porous biomaterial that can bemodified for use based on the disclosure contained in the presentspecification are described in, e.g., Ma, Reverse Fabrication of PorousMaterials, U.S. Pat. No. 6,673,285; Ma, Reverse Fabrication of PorousMaterials, U.S. Patent Publication 2002/0005600; Ratner and Marshall,Novel Porous Materials, U.S. Patent Publication 2008/0075752; Ma,Modified Porous Materials and Method of Forming the Same, U.S. PatentPublication 2008/0213564; Ma, et al., Porous Objects Having ImmobilizedEncapsulated Biomolecules, U.S. Patent Publication 2008/0317816; Hunter,et al., Soft Tissue Implants and Anti-Scarring Agents, U.S. PatentPublication 2009/0214652; Liu, et al., Porous Materials, Methods ofMaking and Uses, Attorney Docket Number 18614 PROV (BRE); and Liu, etal., Porous Materials, Methods of Making and Uses, Attorney DocketNumber 18707PROV (BRE); each of which is incorporated by reference inits entirety.

In one embodiment, a solid hydrogel scaffold can be made as a porousmaterial comprising a hydrogel defined by an interconnected array ofpores. In aspects of this embodiment, pores comprising theinterconnected array of pores have a mean pore diameter in the range of,e.g., about 10 μm to about 1,000 μm, about 200 μm to about 800 μm, about300 μm to about 700 μm, or about 50 μm to about 200 μm.

Alternatively, cells could be blended into a hydrogel formulationcomprising silk fibroin hydrogel particles, carrier solution, or mixtureof silk fibroin hydrogel particles and carrier solution prior toinjection or implantation. Growth factors or other matrix proteins suchas collagen, fibronectin can be added into the silk hydrogels tofacilitate cell growth, differentiation, tissue formation and functionalmatrix deposition. Following implantation, the hydrogel or hydrogelparticles included in the hydrogel formulations comprising cells can beabsorbed into the body over time. This absorption can coincide asinfiltrating tissue replaces the hydrogel material. Thus, a hydrogelscaffold can provide a temporary, well-defined substrate for tissuein-growth during wound healing and soft tissue reconstruction oraugmentation. The disclosed hydrogel scaffolds comprising cells can alsoprovide immediate strength to an incision site or soft tissuereconstruction or augmentation site.

In an embodiment, a hydrogel scaffold comprising cells is bioerodible orbioresorbable. In aspects of this embodiment, a hydrogel scaffoldcomprising cells is bioeroded or bioresorbed, e.g., about 10 days, about20 days, about 30 days, about 40 days, about 50 days, about 60 days,about 70 days, about 80 days, or about 90 days after administration. Inother aspects of this embodiment, a hydrogel scaffold comprising cellsis bioeroded or bioresorbed, e.g., about 10 days or more, about 20 daysor more, about 30 days or more, about 40 days or more, about 50 days ormore, about 60 days or more, about 70 days or more, about 80 days ormore, or about 90 days or more, after administration. In yet otheraspects of this embodiment, a hydrogel scaffold comprising cells isbioeroded or bioresorbed, e.g., about 10 days to about 30 days, about 20days to about 50 days, about 40 days to about 60 days, about 50 days toabout 80 days, or about 60 days to about 90 days after administration.

Aspects of the present specification provide, in part, a cellularcomponent. Cells could be collected from a multitude of hosts includingbut not limited to human autograft tissues, transgenic mammals, orbacterial cultures (possibly for use as a probiotic treatment). Incertain cases, the hydrogel scaffold can comprise human stem cells suchas, e.g., mesenchymal stem cells, synovial derived stem cells, embryonicstem cells, adult stem cells, umbilical cord blood cells, umbilicalWharton's jelly cells, osteocytes, fibroblasts, neuronal cells,lipocytes, bone marrow cells, assorted immunocytes, precursor cellsderived from adipose tissue, bone marrow derived progenitor cells,peripheral blood progenitor cells, stem cells isolated from adult tissueand genetically transformed cells or combinations of the above cells; ordifferentiated cells such as, e.g., muscle cells, adipose cells. Cellsare best added after rinsing of the silk hydrogel material aftergelation. Stem cells can be obtained with minimally invasive proceduresfrom bone marrow, adipose tissue, or other sources in the body, arehighly expandable in culture, and can be readily induced todifferentiate into adipose tissue-forming cells after exposure to awell-established adipogenic inducing supplement. Alternatively, diseasedcells such as cancer cells can be seeded and cultured in silk hydrogels.The seeded silk hydrogel can be used as a model system to study diseasemechanism and to evaluate potential solutions to cure the diseases.Cells can be added to a hydrogel and cultured in vitro to grow tissue oradded to a hydrogel and implanted into a region of the body. The cellscan be seeded on the hydrogel for a short period of time (less than 1day) just prior to implantation, or cultured for a longer (more than 1day) period to allow for cell proliferation and extracellular matrixsynthesis within the seeded matrix prior to implantation.

When utilized as a source of stem cells, adipose tissue can be obtainedby any method known to a person of ordinary skill in the art. Forexample, adipose tissue can be removed from an individual bysuction-assisted lipoplasty, ultrasound-assisted lipoplasty, andexcisional lipectomy. In addition, the procedures can include acombination of such procedures. Suction assisted lipoplasty can bedesirable to remove the adipose tissue from an individual as it providesa minimally invasive method of collecting tissue with minimal potentialfor stem cell damage that can be associated with other techniques, suchas ultrasound assisted lipoplasty. The adipose tissue should becollected in a manner that preserves the viability of the cellularcomponent and that minimizes the likelihood of contamination of thetissue with potentially infectious organisms, such as bacteria and/orviruses.

For some applications preparation of the active cell population canrequire depletion of the mature fat-laden adipocyte component of adiposetissue. This is typically achieved by a series of washing anddisaggregation steps in which the tissue is first rinsed to reduce thepresence of free lipids (released from ruptured adipocytes) andperipheral blood elements (released from blood vessels severed duringtissue harvest), and then disaggregated to free intact adipocytes andother cell populations from the connective tissue matrix. Disaggregationcan be achieved using any conventional techniques or methods, includingmechanical force (mincing or shear forces), enzymatic digestion withsingle or combinatorial proteolytic enzymes, such as collagenase,trypsin, lipase, liberase H1 and pepsin, or a combination of mechanicaland enzymatic methods. For example, the cellular component of the intacttissue fragments can be disaggregated by methods usingcollagenase-mediated dissociation of adipose tissue, similar to themethods for collecting microvascular endothelial cells in adiposetissue, as known to those of skill in the art. Additional methods usingcollagenase that can be used are also known to those of skill in theart. Furthermore, methods can employ a combination of enzymes, such as acombination of collagenase and trypsin or a combination of an enzyme,such as trypsin, and mechanical dissociation.

The active cell population (processed lipoaspirate) can then be obtainedfrom the disaggregated tissue fragments by reducing the presence ofmature adipocytes. Separation of the cells can be achieved by buoyantdensity sedimentation, centrifugation, elutriation, differentialadherence to and elution from solid phase moieties, antibody-mediatedselection, differences in electrical charge; immunomagnetic beads,fluorescence activated cell sorting (FACS), or other means.

Following disaggregation the active cell population can be washed/rinsedto remove additives and/or by-products of the disaggregation process(e.g., collagenase and newly-released free lipid). The active cellpopulation could then be concentrated by centrifugation. In oneembodiment, the cells are concentrated and the collagenase removed bypassing the cell population through a continuous flow spinning membranesystem or the like, such as, for example, the system disclosed in U.S.Pat. No. 5,034,135; and 5,234,608, which are incorporated by referenceherein.

In addition to the foregoing, there are many post-wash methods that canbe applied for further purifying the active cell population. Theseinclude both positive selection (selecting the target cells), negativeselection (selective removal of unwanted cells), or combinationsthereof. In another embodiment the cell pellet could be resuspended,layered over (or under) a fluid material formed into a continuous ordiscontinuous density gradient and placed in a centrifuge for separationof cell populations on the basis of cell density. In a similarembodiment continuous flow approaches such as apheresis and elutriation(with or without counter-current) could be used. Adherence to plasticfollowed by a short period of cell expansion has also been applied inbone marrow-derived adult stem cell populations. This approach usesculture conditions to preferentially expand one population while otherpopulations are either maintained (and thereby reduced by dilution withthe growing selected cells) or lost due to absence of required growthconditions. The active cells that have been concentrated, culturedand/or expanded can be incorporated into disclosed matrices.

In one embodiment, stem cells are harvested, the harvested cells arecontacted with an adipogenic medium for a time sufficient to inducedifferentiation into adipocytes, and the adipocytes are loaded onto abiocompatible matrix which is implanted. In additional embodiments, atleast some of the stem cells can be differentiated into adipocytes sothat a mixture of both cell types is initially present that changes overtime to substantially only adipocytes, with stem cells being present insmall to undetectable quantities. Adipose tissue is fabricated in vivoby the stem cells or prepared ex vivo by the stem cells.

The silk fibroin hydrogels disclosed in the present specification can beused for eye lens replacement. As mentioned above in potentialapplication for vitreous cavity liquid, a firm silk hydrogel with highoptical transparency would be further modified to become bio-inert,potentially through high RGD dosing. In using silk fibroin gels for alens replacement, a silk concentration of about 6% (w/v) to about 10%(w/v) would be most appropriate. This material should be cast in a shapeappropriate for meeting the requirements of a proper lens. AppropriateRGD content for this formulation would be on the order of about 0.0001to about 1 moles of RGD per mole of silk fibroin in the gel material oron the order of about 5 to about 100 moles of RGD per mole of silkfibroin in the gel. A small RGD component or very large RGD componentcould both be very useful in discouraging cell proliferation eitherthrough denial of binding motifs or through provision of so many as toanchor cells and deny the ability to move and proliferate.

The silk fibroin hydrogels disclosed in the present specification can beused as a platform for drug delivery. For example, the silk hydrogel canbe formed with a pharmaceutical agent either entrained in or bound tothe gel and injected, implanted, or delivered orally into the body. Forextended or sustained-drug delivery, silk hydrogel can manipulated to behighly resistant to bioresorption and hydrophobic under certainconditions (e.g., very high β-sheet content) which discouragescell/tissue ingrowth. This in turn leads to prolonged gel bioresorptionand, by extension, prolonged drug release such as, e.g., sustained drugrelease or extended drug release. The silk fibroin hydrogel platform canbe produced as a gel or as a solid. The pharmaceutically-active drug canbe, without limitation, proteins, peptides, steroids, antibiotics,vitamins, simple sugars, genes, transfected or non-transfected cells. Tocontrol the drug release profile, the pharmaceutically-active drug canbe first mixed with silk solutions then form a hydrogel. This silkhydrogel can then be ground into smaller particles mixed with anadditional silk gel phase acting as a carrier either with or without aviscosity inducing component, a surfactant, and/or an included lubricantfluid like saline. The therapeutic-bound silk hydrogel can also befurther crosslinked to enhance the stability to extend the releaseperiod. In an alternative approach, silk hydrogel can be mixed withother polymers, for examples, hyaluronic acid, to prolong the release ofcertain growth factors or cytokines and to stabilize the functionality.Furthermore, the silk fibroin hydrogel can serve as the outer coatingfor coaxial drug delivery systems.

Aspects of a drug delivery platform comprising the silk fibroinhydrogels disclosed in the present specification are sustained releasedrug delivery platforms. As used herein, the term “sustained release”refers to the release of a pharmaceutically-active drug over a period ofabout seven days or more. In aspects of this embodiment, a drug deliveryplatform comprising the silk fibroin hydrogel releases apharmaceutically-active drug with substantially first order releasekinetics over a period of, e.g., about 7 days after administration,about 15 days after administration, about 30 days after administration,about 45 days after administration, about 60 days after administration,about 75 days after administration, or about 90 days afteradministration. In other aspects of this embodiment, a drug deliveryplatform comprising the silk fibroin hydrogel releases apharmaceutically-active drug with substantially first order releasekinetics over a period of, e.g., at least 7 days after administration,at least 15 days after administration, at least 30 days afteradministration, at least 45 days after administration, at least 60 daysafter administration, at least 75 days after administration, or at least90 days after administration.

Aspects of a drug delivery platform comprising the silk fibroinhydrogels disclosed in the present specification are extended releasedrug delivery platforms. As used herein, the term “extended release”refers to the release of a pharmaceutically-active drug over a period oftime of less than about seven days. In aspects of this embodiment, adrug delivery platform comprising the silk fibroin hydrogel releases apharmaceutically-active drug with substantially first order releasekinetics over a period of, e.g., about 1 day after administration, about2 days after administration, about 3 days after administration, about 4days after administration, about 5 days after administration, or about 6days after administration. In other aspects of this embodiment, a drugdelivery platform comprising the silk fibroin hydrogel releases apharmaceutically-active drug with substantially first order releasekinetics over a period of, e.g., at most 1 day after administration, atmost 2 days after administration, at most 3 days after administration,at most 4 days after administration, at most 5 days afteradministration, or at most 6 days after administration.

Aspects of a drug delivery platform comprising the silk fibroinhydrogels disclosed in the present specification are extended releasedrug delivery platforms. As used herein, the term“pharmaceutically-active drug” refers to a substance used in thediagnosis, treatment, or prevention of a disease or as a component of amedication.

Aspects of a drug delivery platform comprising the silk fibroinhydrogels disclosed in the present specification may, or may not,further comprise a solubilizing component. The use of such asolubilizing component is advantageous to provide any relatively quickrelease of the pharmaceutically-active drug into the body fortherapeutic effectiveness. Such solubilizing component, of course,should be physiologically-acceptable. In one embodiment of the presentdrug delivery platform, an effective amount of a solubilizing componentis provided to solubilize a minor amount, that is less than 50%, forexample in a range of about 1% or about 5% to about 10% or about 20% ofthe pharmaceutically-active drug. For example, the inclusion of acyclodextrin component, such as β-cyclodextrin, sulfo-butyletherβ-cyclodextrin (SBE), other cyclodextrins and the like and mixturesthereof, at about 0.5 to about 5.0% (w/v) solubilizes about 1% to about10% of the initial dose of a pharmaceutically-active drug. Thispresolubilized fraction provides a readily bioavailable loading dose,thereby avoiding any delay time in therapeutic effectiveness.

Aspects of a drug delivery platform comprising the silk fibroinhydrogels disclosed in the present specification may, or may not,further comprise a sustained release component. Sustained releasecomponents, include, without limitation, polymers (in the form forexample of gels and microspheres), such as, e.g., poly(D,L-lactide) orpoly(D,L-lactide co-glycolide), in amounts effective to reduce localdiffusion rates and/or corticosteroid particle dissolution rates. Theresult is a flatter elimination rate profile with a lower C_(max) and amore prolonged therapeutic window, thereby extending the time betweenrequired injections for many patients. Any suitable, preferablyconditionally acceptable, release component may be employed. Thesustained release component is preferably biodegradable or bioabsorbablein the eye so that no residue remains over the long term. The amount ofthe delayed release component included may very over a relatively widerange depending, for example, on the specific sustained releasecomponent is being employed, the specific release profile desired andthe like factors. Typical amounts of delayed release components, if any,included in the present compositions are in a range of about 0.05% (w/v)to 0.1% (w/v) to about 0.5% (w/v) or about 1% (w/v) or more (weight ofthe ingredient in the total volume of the composition) of thecomposition.

The silk fibroin hydrogels disclosed in the present specification can beused as a surface coating to improve the functionality of medicaldevices. For example, the silk hydrogel can be formed on the surface ofa silk yarn or mesh to improve the biocompatibility of the device. Thevolume of the silk hydrogel may be gradually replaced by ingrown tissue.This silk hydrogel surface coating may contain pharmaceutical agentssuch as growth factors to help to realize the desired outcome. Forexample, PDGF can be added to improve ligament and tendon regeneration,TGF-β can be added to improve cartilage regeneration, and antibioticscan be added to cure or prevent infection at the implantation site. Useof drugs such as local anesthetics, lidocaine for example, inconjunction with the silk gel could reduce the pain caused by injectionsof the material. The device can cast and molded or cast and reshaped,then implanted for the tissue reconstruction. In addition, the silkfibroin gel could be combined with cells for purposes of a biologicallyenhanced repair.

Silk gel could be employed as a coating in a number of differentfashions. Devices could be soaked in silk solution, and then exposed toaccelerant in a mold such that the silk gel would be developed in aspecific shape according to the mold. In another embodiment, the devicecould be soaked in silk solution, and then exposed to a bulk ofaccelerant through a dipping process, yielding a uniform layer of silkgel coating the device. In another embodiment, it would be possible forthe device to be soaked in accelerant and then introduced into a bulk ofsilk solution, again forming a uniform layer of silk gel coating thedevice. In another embodiment, the device could be dipped into a mixtureof gelling silk solution and accelerant. In another embodiment, agelling mixture of silk solution and accelerant could be added to thedevice in a mold. In yet another embodiment, the gel components could becast separately onto the device by, e.g., spraying or knifing, and thisapplication would mix the components, thereby causing gelation. In anyof these cases, a therapeutic agent could be added as describedpreviously. Conventional techniques for modifying surface texture couldalso be applied here such as introduction and dissolution of crystallinesalts.

The silk concentration of the coating would be determined based upon theprojected need for the device itself. In the case of a hard coating, asilk fibroin concentration of about 6% (w/v) to about 10% (w/v) would beappropriate. In the case of a soft coating, about 2% (w/v) to about 5%(w/v) would be appropriate. The RGD concentration of the coating wouldbe determined based upon the desired biological performance of thedevice. For general tissue interaction and ingrowth, a concentration ofRGD between about 0.1 and about 3 moles of RGD per mole of silk would beappropriate. In cases where a muted host ingrowth profile weredesirable, appropriate RGD content would be on the order of about 0.0001to about 0.01 moles of RGD per mole of silk in the gel material or onthe order of about 5 to about 100 moles of RGD per mole of silk in thegel. A small RGD component or very large RGD component could both bevery useful in discouraging cell proliferation either through denial ofbinding motifs or through provision of so many as to anchor cells anddeny the ability to move and proliferate.

In one embodiment, the present invention provides a five-amino acidpeptide “tail” capable of linking or conjugating a molecule X to a silkmolecule or fibroin when the molecule X is attached to the tail. As usedherein, the term “linking” or “conjugating” in the context of molecule Xrefers to an indirect physical attachment of a molecule X to a silkfibroin via a third entity, the five-amino acid peptide “tail” beingthat entity. In one embodiment, the tail binds to silk fibroin byhydrophobic interaction to the silk fibroin. Alternatively, the “tail”binds the silk molecules by hydrogen bonding and/or covalent bonding. Itis envisioned that the “tail” can bind silk fibroins by a combination ofhydrophobic interactions, hydrogen bonds, and covalent bonds. Byattaching a molecule X to a “tail” described herein, it is possible toindirectly link the molecule X to silk fibroin via the tail, and thus tothe silk hydrogels described herein. Accordingly, in one embodiment, thefive-amino acid peptide “tail” comprises hydrophobic and/or apolar (nonpolar) amino acid residues such as valine, leucine, isoleucine,phenylalanine, tryptophan, methionine, cysteine, alanine, tyrosine,serine, proline, histidine, threonine and glycine. Various combinationsof hydrophobic and/or apolar amino acid residues are possible, for e.g.LLLLL (SEQ ID NO: 15), LLFFL (SEQ ID NO: 16), LFLWL (SEQ ID NO: 17),FLWLL (SEQ ID NO: 18) and LALGL (SEQ ID NO: 19). In other embodiments,the tail comprises any combination of the twenty standard conventionalamino acid residues. In other embodiments, the tail compriseshydrophobic and/or apolar (non polar) and amino acids residues withhydrophobic side chains, e.g. arginine and lysine. As used herein, theterm “comprising” or “comprises” means that other elements can also bepresent in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

In one embodiment, the molecule X is attached to a tail at the carboxyl(COOH) end of the five-amino acid peptide. In another embodiment, themolecule X is attached to a tail at the amino (NH₂) end of thefive-amino acid peptide.

In one embodiment, the five-amino acid peptide “tail” capable of linkingor conjugating a molecule X to a silk molecule or fibroin when themolecule X is attached to the tail comprise more than five amino acidresidues, e.g. six or seven hydrophobic and/or apolar amino acidresidues, such as LLLLLL (SEQ ID NO: 20).

In one embodiment, the five-amino acid peptide “tail” comprises aminoacid residues that are part hydrophobic (i.e. the part of the side-chainnearest to the protein main-chain), for e.g. arginine and lysine. In oneembodiment, the part hydrophobic amino acid residues flank thefive-amino acid peptide “tail” such as in RLLLLLR (SEQ ID NO: 21),KLLLLLR (SEQ ID NO: 22) and KLLLLLK (SEQ ID NO: 23).

In one embodiment, the five-amino acid peptide “tail” is separated froma molecule X by a spacer peptide. Spacer peptides should generally havenon-polar amino acid residues, such as, glycine and proline. In oneembodiment, the spacer comprises unnatural amino acid residues such asnor amino acids and keto-substituted amino acids. Such unnatural aminoacid residues are well known to one skilled in the art.

In one embodiment, the spacer peptide is attached to a tail at thecarboxyl (COOH) end of the five-amino acid peptide. In anotherembodiment, the spacer is attached to a tail at the amino (NH₂) end ofthe five-amino acid peptide.

The length of the space peptide is variable. The spacer serves to linkthe molecule X and tail together and also to provide steric freedom tothe molecule X, allowing for proper orientation of a molecule X (e.g.cell binding domains such as the RGD domain) and the correct interactionof the molecule X with cells in vivo. A spacer which is too short canprevent the molecule X from being properly functional (i.e., holding ittoo tight to the silk molecules and away from cells), a spacer which istoo long can cause undesired effects as well (i.e., non-specificassociation of peptides or shortened efficacy from peptide due to spacerbreakage). In one embodiment, the number of amino acid residues in aspacer can range from 1 to 300. In one embodiment, the spacer comprisesa single amino acid residue, such as a G or a P. Examples of spacerswith more amino acid residues are GSPGISGGGGGILE (SEQ ID NO: 24) andSGGGGKSSAPI (SEQ ID NO: 25).

In one embodiment, the molecule X is any biological molecule or fragmentthereof. Examples biological molecules include but are not limited togrowth factors, hormones, cytokines, chemokines, extracellular matrixcompounds, osteogenic protein (OP), bone morphogenetic protein (BMP),growth and differentiation factor (GDF), transforming growth factor(TGF), epidermal growth factor (EGF), vascular endothelial growth factor(VEGF), interleukin (IL), platelet derived growth factor (PDGF),fibroblast growth factor (FGF), insulin-like growth factor (IGF), basicfibroblast growth factor (BFGF), fibroblast activation protein (FAP),disintegrin, metalloproteinase (ADAM), matrix metalloproteinase (MMP),connective tissue growth factor (CTGF), stromal derived growth factor(SDGF), keratinocyte growth factor (KGF), tumor necrosis factor (TNF),interferon (IFN), erythropoietin (EPO), hepatocyte growth factor (HGF),thrombopoietin (TPO), granulocyte colony stimulating factor (GCSF),granulocyte macrophage colony stimulating factor (GMCSF), myostatin(GDF-8), collagen, elastin, laminin, hyaluronic acid, decorin, actin,and tubulin. Examples fragments of biological molecules include but arenot limited to known cell integrin binding domains including but notlimited to RGD, KQAGDV (SEQ ID NO: 4), PHSRN (SEQ ID NO: 5), YIGSR (SEQID NO: 6), CDPGYIGSR (SEQ ID NO: 7), IKVAV (SEQ ID NO: 8), RNIAEIIKDI(SEQ ID NO: 9), YFQRYLI (SEQ ID NO: 10), PDSGR (SEQ ID NO: 11), FHRRIKA(SEQ ID NO: 12), PRRARV (SEQ ID NO: 13), and WQPPRARI (SEQ ID NO: 14).

In other embodiments, the molecule X is any recombinant, synthetic, ornon-native polymeric compounds. Examples include but are not limited tochitin, poly-lactic acid (PLA), poly-glycolic acid (PGA), as tracers(e.g. radioisotopes), contrasting agents (e.g. imaging dyes), aptamers,avimers, peptides, nucleic acids, modified polysaccharide coatings,drugs (chemotherapy drugs), and recombinant antibodies or antibody-basedmoieties.

In one embodiment, the present invention provides a synthetic moleculehaving the formula: (molecule X)_(n)-(spacer peptide)₀₋₃₀₀-(tail)-NH₂for linking with silk molecule or fibroin, wherein “n” is a wholeinteger ranging from 1-30, and wherein the amino acid residues of thespacer ranges from 0-300. Examples of such synthetic molecule capablefor linking to silk molecule or fibroin are: GRGDIPASSKG₄SRL₆R-NH₂ (SEQID NO: 1), Ac-GdRGDIPASSKG₄SdRL₆dR-NH₂ (SEQ ID NO: 2),(VEGF)-(VEGF)-GSPGISGGGGGILEKLLLLLK-NH₂ (SEQ ID NO: 26),(HIV-C-peptide)₃-GSPGISGGGGGILEKLALWLLR-NH₂ (SEQ ID NO: 27),(taxol)₂-GSPGISGGGGGILERLLLLR-NH₂ (SEQ ID NO: 28), and(EPO)₂-GSPGISGGGGGILERLLWLLR-NH₂ (SEQ ID NO: 29). When used in thecontext of the silk hydrogel described herein, the synthetic molecule ofSEQ ID NO: 1 enable better tissue attachment of the hydrogel constructin vivo, the synthetic molecule of SEQ ID NO: 26 can promote bloodvessel generation (neo-angiogenesis) in tissue engineered constructs,the synthetic molecule of SEQ ID NO: 28 can provide a slow releaseanti-HIV medication in the form of a transdermal delivery patch, thesynthetic molecule of SEQ ID. NO: 28 can provide sustained dosage ofanti-cancer drug in vivo, and the synthetic molecule of SEQ ID NO: 29can provide a slow release EPO during cancer chemotherapy treatment.

Encompassed in the invention are injectable silk hydrogel formulationscomprising a synthetic molecule having the formula: (moleculeX)_(n)-(spacer peptide)₀₋₃₀₀-(tail)-NH₂ or a synthetic molecule havingthe formula: (molecule X)_(n)-(spacer peptide)₀₋₃₀₀-(tail)-NH₂ and anamphiphilic peptide. The amphiphilic peptide is 23RGD.

Basically, a molecule X is any entity, natural or synthetic, that can beuseful and can be use in the context of silk hydrogels.

In one embodiment, the invention provides a method of conjugating amolecule X to a silk molecule or fibroin comprising mixing a syntheticmolecule having the formula: (molecule X)_(n)-(spacerpeptide)₀₋₃₀₀-(tail)-NH₂ with a silk molecule or fibroin or silksolution.

Methods of peptide synthesis are known to one skilled in the art, forexample, the peptides described herein can be synthetically constructedby suitable known peptide polymerization techniques, such as exclusivelysolid phase techniques, partial solid-phase techniques, fragmentcondensation or classical solution couplings. For example, the peptidesof the invention can be synthesized by the solid phase method usingstandard methods based on either t-butyloxycarbonyl (BOC) or9-fluorenylmethoxy-carbonyl (FMOC) protecting groups. This methodologyis described by G. B. Fields et al. in Synthetic Peptides: A User'sGuide, W. M. Freeman & Company, New York, N.Y., pp. 77-183 (1992) and inthe textbook “Solid-Phase Synthesis”, Stewart & Young, Freemen &Company, San Francisco, 1969, and are exemplified by the disclosure ofU.S. Pat. No. 4,105,603, issued Aug. 8, 1979. Classical solutionsynthesis is described in detail in “Methoden der Organischen Chemic(Houben-Weyl): Synthese von Peptiden”, E. Wunsch (editor) (1974) GeorgThieme Verlag, Stuttgart West Germany. The fragment condensation methodof synthesis is exemplified in U.S. Pat. No. 3,972,859. Other availablesyntheses are exemplified in U.S. Pat. No. 3,842,067, U.S. Pat. No.3,872,925, issued Jan. 28, 1975, Merrifield B, Protein Science (1996),5: 1947-1951; The chemical synthesis of proteins; Mutter M, Int J PeptProtein Res 1979 March; 13 (3): 274-7 Studies on the coupling rates inliquid-phase peptide synthesis using competition experiments; and SolidPhase Peptide Synthesis in the series Methods in Enzymology (Fields, G.B. (1997) Solid-Phase Peptide Synthesis. Academic Press, San Diego.#9830). The foregoing disclosures are incorporated herein by reference.Molecular DNA methods can also be used. The coding sequence of the shortspacer can be constructed be annealing a complementary pair of primers.One of skill in the art can design and synthesize oligonucleotides thatwill code for the selected spacer.

Methods of linking peptides are also known in the art. The physicallinking of the individual isolated peptides into oligomeric peptides asset forth herein, can be effected by chemical conjugation procedureswell known in the art, such as by creating peptide linkages, use ofcondensation agents, and by employing well known bifunctionalcross-linking reagents. The conjugation may be direct, which includeslinkages not involving any intervening group, e.g., direct peptidelinkages, or indirect, wherein the linkage contains an interveningmoiety, such as a protein or peptide, e.g., plasma albumin, or otherspacer molecule. For example, the linkage may be via aheterobifunctional or homobifunctional cross-linker, e.g., carbodiimide,glutaraldehyde, N-succinimidyl 3-(2-pyridydithio) propionate (SPDP) andderivatives, bis-maleimide,4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and the like.

Cross-linking can also be accomplished without exogenous cross-linkersby utilizing reactive groups on the molecules being conjugated. Methodsfor chemically cross-linking peptide molecules are generally known inthe art, and a number of hetero- and homobifunctional agents aredescribed in, e.g., U.S. Pat. Nos. 4,355,023, 4,657,853, 4,676,980,4,925,921, and 4,970,156, and Immuno Technology Catalogue and Handbook,Pierce Chemical Co. (1989), each of which is incorporated herein byreference. Such conjugation, including cross-linking, should beperformed so as not to substantially affect the desired function of thepeptide oligomer or entity conjugated thereto, including therapeuticagents, and moieties capable of binding substances of interest.

Conjugation of individual peptide can be effected by a linkage via theN-terminal or the C-terminal of the peptide, resulting in an N-linkedpeptide oligomer or a C-linked peptide oligomer, respectively.

It will be apparent to one skilled in the art that alternative linkerscan be used to link peptides, for example the use of chemical proteincrosslinkers. For example homobifunctional crosslinker such asdisuccinimidyl-suberimidate-dihydrochloride;dimethyl-adipimidate-dihydrochloride; 1,5,-2,4-dinitrobenzene orheterobifunctional crosslinkers such as N-hydroxysuccinimidyl2,3-dibromopropionate; 1ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride; andsuccinimidyl4-[n-maleimidomethyl]-cyclohexane-1-carboxylate.

Several embodiments will now be described further by non-limitingexamples.

EXAMPLES

The following examples illustrate representative embodiments nowcontemplated, but should not be construed to limit the disclosedpurified silk fibroin and method for purifying such silk fibroins,hydrogels comprising such silk fibroin with or without an amphiphilicpeptide and methods for making hydrogels comprising such silk fibroinand the use of silk fibroin hydrogels in a variety of medical uses,including, without limitation fillers for tissue space, templates fortissue reconstruction or regeneration, scaffolds for cells in tissueengineering applications and for disease models, a surface coating toimprove medical device function, or as a platform for drug delivery.

Example 1 Silk Sericin Extraction

Silk fibroin for generation of the hydrogel was obtained in the form ofdegummed B. mori silk at a size of 20 denier-22 denier (38 μm±5.6 μmdiameter). This degummed silk was further processed in order to removethe inherently present and potentially antigenic protein glue, sericinthat conjoins independent fibroin filaments. This was done as describedpreviously herein. Following removal of sericin, the pure fibroin wasdried carefully to ambient humidity levels using a laminar flow hood.

Example 2 Generation of Silk Fibroin Solution

Silk fibroin filaments, cleaned of their sericin and rinsed free ofinsoluble debris and ionic contaminants were used for the generation ofan aqueous silk solution. These silk fibers were added to a solution of9.3M LiBr and purified water (e.g., MILLI-Q® Ultrapure WaterPurification Systems) (Millipore, Billerica, Mass.) to make a solutionconsisting of 20% pure silk (% w/v). This mixture was then heated to atemperature of 60° C. and digested for a period of four hours. A totalof 12 mL of the resultant solution was then loaded into a 3 mL-12 mLSlide-A-Lyzer dialysis cassette (Pierce Biotechnology, Inc., Rockford,Ill.) (molecular weight cutoff of 3.5 kD) and placed into a beakercontaining purified water as a dialysis buffer at a volume of 1 L waterper 12 mL cassette of silk solution. The beakers were placed on stirplates and stirred continuously for the duration of the dialysis.Changes of dialysis buffer occurred at 1, 4, 12, 24, and 36 hours ofprocessing time.

Following dialysis, the solution was removed from the cassettes by meansof a syringe and needle and centrifuged at 30,000 g relative centrifugalforce (RCF) at 4° C. for 30 minutes, decanting the supernatant (silksolution) into a clean centrifuge tube, then repeating thecentrifugation for a further 30 minutes. This process of centrifugationis beneficial for removal of insoluble particulate debris associatedwith the silk solution both prior to and following after dialysis. It isbelieved that such insoluble debris could serve as antigens in vivo orperhaps nucleation points about which gelation of the silk could occur,shortening storage life of the solution and compromising the uniformityof the gelation system. After completion of the second centrifugation,the supernatant was again collected and stored at 4° C. until needed. Toconfirm uniformity of the dialysis product, known volumes of thesolution were collected, massed, and then dried completely throughlyophilization. These lyophilized samples were then massed and the drymass of solution compared to initial solution volume to determinepercent silk present per unit volume of solution. Additionally, thesolution was assessed via X-ray Photoelectron Spectroscopy (XPS)analysis to ensure that no detectable quantities of Li⁺ or Br⁻ ions werepresent in the solution.

Example 3 Induction of Gelation

A variety of different methods were employed in the course of hydrogeldevelopment for the purposes of contrasting and comparing certainrelevant properties of various formulae. Regardless of the nature inwhich the gelation process was carried out, the final determination thata “gel” state had been reached was applied uniformly to all groups. Asolution or composite of solutions (i.e., silk solution blended with anenhancer or enhancer solution) was considered a gel after observingformation of a uniform solid phase throughout the entire volume,generally opaque and white in appearance.

Samples to be produced by passive gelation were not exposed to anyenhancer additives. These gels were produced by measuring a volume ofsilk solution into a casting vessel, for the purposes of theseexperiments, polypropylene tubes sealed against air penetration andwater loss, and the sample allowed to stand under ambient roomconditions (nominally 20-24° C., 1 atm, 40% relative humidity) untilfully gelled. Care was taken to ensure uniformity of casting vesselsmaterial of construction across groups so as to avoid potentialinfluence from surface effects. These effects may serve to enhance orinhibit gelation and may be caused by factors including but not limitedto siliconization, surface roughness, surface charge, debriscontamination, surface hydrophobicity/hydrophilicity, and altered masstransfer dynamics.

Samples produced by means of a 23RGD-induced process were made in one oftwo ways, the first being direct addition of 23RGD in a pre-determinedratio to the silk solution without any sort of reconstitution. The 23RGD(obtained as a desiccated fine powder form) was blended into a measuredvolume of 8% silk solution within the casting vessel by pipetting usinga 1000 μL pipette. These gels were then cast in polypropylene tubes,sealed against air penetration and water loss, and the sample wasallowed to stand under ambient room conditions (nominally 20-24° C., 1atm, 40% relative humidity) until fully gelled.

The 23RGD-induced gels were also produced by first dissolving the 23RGDpowder in purified water. The concentration of this solution wasdetermined based upon the amount of 23RGD to be introduced into a geland the final concentration of silk desired in the gel. In the case of4% silk gels enhanced with 23RGD, quantities of water equal to theamount of 8% silk solution to be used in the gel were used for thedissolution of appropriate quantities of 23RGD. In the case of gelsinduced by addition of 23RGD to be generated at a molar ratio of 3:123RGD:silk, a quantity of 23RGD was dissolved in 1 mL of water per 1 mLof 8% silk solution to be gelled. This mixing was performed in thecasting vessel as well, being accomplished by means of rapid pipettingwith a 1000 μL pipette when appropriate. These gels were then cast inpolypropylene tubes, sealed against air penetration and water loss, andthe sample was allowed to stand under ambient room conditions (nominally20-24° C., 1 atm, 40% relative humidity) until fully gelled.

Samples produced by means of ethanol-enhanced gelation (EEG) weregenerated by means of directly adding ethanol to a measured volume of 8%silk solution in the casting vessel. The ethanol is added in a quantitysuch that the volume added should yield a volumetric dilution of the 8%silk solution resulting in the final required concentration of silkwithin the gel, assuming minimal volume loss due to miscibility of theorganic added. The mixture of ethanol and silk solution is then mixed bymeans of pipetting with a 1000 μL pipette when appropriate. These gelswere then cast in polypropylene tubes, sealed against air penetrationand water loss, and the sample was allowed to stand under ambient roomconditions (nominally 20-24° C., 1 atm, 40% relative humidity) untilfully gelled.

Samples produced by a combined 23RGD-ethanol effect (RGDEEG) weregenerated using a solution of 90% ethanol, 10% purified water andappropriate quantities of 23RGD dissolved in this solvent. It was notpossible to readily dissolve 23RGD in pure ethanol and it was believedthat undissolved 23RGD might cause poor distribution of the peptidethroughout the gel phase. As a result, it was determined that since asolution of ethanol and water offering similar gelation accelerationcharacteristics to a pure ethanol solution and reasonable 23RGDsolubility would be an acceptable alternative. A solution of 90% ethanoland 10% water met both of these criteria and as a result was used forgeneration of these gels. The 23RGD concentration of this ethanolsolution was determined based upon the amount of 23RGD to be introducedinto a gel and the final concentration of silk desired in the gel. Inthe case of 4% silk gels enhanced with 23RGD, quantities of 90% ethanolequal to the amount of 8% silk solution to be used in the gel were usedfor the dissolution of appropriate quantities of 23RGD. In the case ofgels induced by addition of 23RGD to be generated at a molar ratio of3:1 23RGD:silk, a quantity of 23RGD was dissolved in 1 mL of 90& ethanolper 1 mL of 8% silk solution to be gelled. This mixing was performed inthe casting vessel as well, being accomplished by means of rapidpipetting with a 1000 μL pipette when appropriate. These gels were thencast in polypropylene tubes, sealed against air penetration and waterloss, and the sample was allowed to stand under ambient room conditions(nominally 20-24° C., 1 atm, 40% relative humidity) until fully gelled.

Silk gelation times were determined by casting gels according to themethods above, the exception being that gels were mixed not throughpipetting, but through vigorous mechanical shaking. These studies wereconducted using 1.5 mL microcentrifuge tubes as casting vessels withsample groups of N=6 used for each gel formulation (FIG. 1). Thedetermination that a “gel” state had been reached was made in the methodas described above, based upon observation of a uniform solid phasethroughout the entire volume, generally opaque and white in appearance.

Gelation time varied widely depending on specific formulation. The 8Psilk samples took 21 days until gelation while the 4P samples required31±1 day (data not shown). EEG samples gelled significantly faster thanPG samples with a 4E sample requiring 27±5.4 seconds for gelation (p0.05). EEG samples gelled more rapidly as the concentration of ethanoladded increased with time required gelation times of 1770±600 s,670.3±101.0 s, 29.8±5.2 s, 9.7±2.0 s, and 4.2±0.8 s for 6.4E, 6E, 4.8E,4E, and 3.2E respectively. There were significant differences betweenall times except 4.8E and 4E, 4E and 3.2E, and 4.8E and 3.2E. RGDEEGgels generated a tightly localized white fibrous precipitateinstantaneously upon addition of the ethanol solution to the silk andgelled more quickly than PG samples, though they were slower than EEGgels. 4RL, 4RM and 4RH samples took 22.7±2.5 seconds, 38.8±4.5 seconds,and 154.5±5 seconds to gel with 4RH differing significantly from theother RGDEEG formulations.

Gelation timing experiments revealed the time constraints posed by thePG method. Results indicated that, while increased silk concentrationdecreased gelation time, the total time to gel was decreased only from31 days for 4P to 21 days for 8P. This may result from the increasedfrequency of collisions between silk molecules in solution and resultantgel network assembly. Using ethanol directly added to silk solution asan accelerant proved to dramatically decrease the gelation time of thesilk by increasing the volume of ethanol added in a fashion well-modeledby a power function. This increasingly rapid gelation is likely causedby greater competition for hydrating water molecules between silk andethanol coupled with altered electronegativity of the solution, bothfavoring forced aggregation of the silk molecules. Studies conducted onRGDEEG samples revealed that addition of greater concentrations of RGDled to increasing gelation times modeled by an exponential function.This appears counter-intuitive as it was expected that RGD shouldfunction in some capacity to accelerate gelation.

The slowing of gelation in RGDEEG samples may result from difficultiesin silk molecular binding to the RGD-coated silk precipitates, perhapsdue to stearic interference with hydrophobic regions of silk chains.Upon RGD-ethanol accelerant addition to the silk solution, a largequantity of silk-RGD complexes was precipitated from the solution. Itwas noted during the gelation of RGDEEG samples that a fibrillar, white,opaque precipitate was consistently formed within the solution mixtureimmediately upon mixing. This precipitation from solution may beevidence of this rapid assembly of high concentration silk-RGDprecipitates. This formation may be caused by association between silkmicelles and peptide molecules in solution, disruption of the silkmicelles, and rapid assembly of them into a tightly-localized fibrillarstructure. This rapid assembly may progress until driving gradientsgenerated by the differing solvent chemistries provided by the ethanoland water reach an equilibrium state. At this point, silk molecules areable to remain stably in solution with further silk network assemblyoccurring only by slow lengthening of the initially formed precipitates.While this precipitation provided a high number of nucleation points toinitiate completion of a gel network, these nucleation points may be oflimited utility based upon availability of binding sites. The remainingsilk molecules were much slower to assemble as a result. Theseprecipitates also tended to initiate assembly of a peripheral networkcomprised largely of loose α-helix and random coil motifs, possibly dueto interference in silk packing due to the interference of theseparticles.

The hydrogels produced by the methods described above derive substantialbenefit from the ability to more precisely control the time course forits gelation in comparison to that of a conventionally designed and castgel. It is evident from monitoring the time between casting and gelationof the device and similarly cast, non-enhanced or exclusively ethanolmodified gels that 23RGD under certain circumstances may be manipulatedto have an additional accelerant effect upon the process of gelation.This observed enhancer effect both mitigates the time constraints andcontrollability associated with non-modified gels and additionallyalters the manner in which the protein aggregate network is formedrelative to solely ethanol enhanced gels.

Example 4 Determination of Residual Ethanol by Colorimetric Analysis

Following gelation of a sample produced with either an ethanol or 23RGDcomponent, the gel was removed from the casting vessel and immersed in abulk of purified water as a rinse buffer. This bulk comprised a volumesuch that the volumetric ratio of water to gel was ≧100:1. The gel waspermitted to lay static in the rinse buffer for a period of 72 hours,changing the water every 12 hours.

Samples of silk gel were evaluated to determine the total residualcontent of ethanol in a series of 23RGD-ethanol- and ethanol-enhancedgels. Briefly, samples of gel (N=4 of each type) generated as describedabove were processed and analyzed using an Ethanol Assay Kit (kit #K620-100 from BioVision Research Prods, Mountain View, Calif.). Samplesof gel were cut to a size of approximately 0.3 cm in height by 0.5 cm indiameter (approximately 250 mg). These samples were massed to thenearest 0.1 mg using an APX-60 (Denver Instrument, Denver Colo.) balanceas per the manufacturer's instructions. These gel samples wereindividually ground using a metal spatula and placed into 250 μL ofMilli-Q water in microcentrifuge tubes. These gels were incubated at 37°C. for a period of 24 hours. After incubation, the gels were centrifugedon an Eppendorf 5415 microcentrifuge with an HA 45-18-11 rotor (Hamburg,Germany) at 18,000 rpm for 30 minutes. At the conclusion of thiscentrifugation step, the supernatant was used as the sample of interestaccording to the instructions provided by the kit manufacturer.Colorimetric analyses of the sample was performed at an absorbance of570 nm using a spectrophotometer, and in conjunction with a standardcurve, residual percentages of ethanol in the gel were calculated (Table1, FIG. 2). It was shown in this process that the leeching step iscapable of substantially removing residual ethanol from the silk gels,as none of these materials exhibited a residual ethanol component ofgreater than 5% ethanol by mass.

TABLE 1 Determination of Residual Ethanol by Colorimetric Analysis SilkInitial Final Ethanol Concen- Enhancer Enhancer Ethanol Concentrationtration Solvent Solute Concentration Mean Stdev 2% 90% None 68% 2.49%0.06% 3:1 4.44% 0.13% 23RDG:Silk 10:1 4.77% 0.29% 23RDG:Silk 4% None 45%2.55% 0.07% 3:1 2.86% 0.08% 23RDG:Silk 10:1 2.97% 0.07% 23RDG:Silk 6%None 22.5%  3.12% 0.05% 3:1 3.16% 0.04% 23RDG:Silk 10:1 2.99% 0.10%23RDG:Silk

Example 5 23RGD Quantification by HPLC

23RGD-infused gels were studied to quantify the amount of 23RGD bound tothe silk-hydrogel device as well as the quantity of free 23RGD whichmight be rinsed free of the device under relevant conditions. Briefly,samples of 23RGD-infused gel were cast and rinsed according to themethods above, with samples of rinse buffer being collected from eachrinse for subsequent analysis by HPLC. Additionally, subsequent to thelast rinse, the gel samples were mechanically pulverized by means of astainless steel stirring rod and the adsorbed 23RGD removed byincubation for 4 hours in a dissolving buffer. This mixture of gel andsolvent was then centrifuged on an Eppendorf 5415C at 16,000 g RCF for30 minutes. The supernatant was collected and centrifuged another 30minutes at 16,000 g RCF after which time the supernatant was collectedin a sample vial for HPLC analysis. Samples of rinse buffer from thefirst and last rinse were centrifuged in the same fashion after beingdiluted with the same solvent the gel was extracted with in a volumetricratio of 1 part rinse buffer to 4 parts solvent. To ensure23RGD-hydrogel device rinse-exposed surface area was not a limitingfactor, the same rinse and extraction process was performed upon devicespulverized after gelation and before rinsing. The peak area consistentwith 23RGD for each HPLC sample was taken and these data comparedagainst a standard curve generated for 23RGD on the same HPLC unit underidentical handling and run conditions.

The resultant data indicated levels of signal from 23RGD in samplescollected from rinse buffer were just slightly higher than values for23RGD solvent alone and were immeasurable by the standard curve,expected to resolve a relative 23RGD:silk ratio of 0.05:1. Bycomparison, the assay was able to detect a ratio of 3.35:1 in the finalrinsed and extracted 23RGD-enhanced gel.

HPLC data confirmed complete retention of RGD on the silk hydrogelmaterial after the rinse process. This provides not only a functionalRGD component to this specific series of hydrogel formulations, butindication for use of amphiphilic peptides as candidates forintroduction of other components into silk gels. This knowledge might beapplied to a number of other biologically active peptide sequences,though additional work must be done to understand how these specificpeptides might influence gelation and how gelation in turn impacts thefunctionality of these peptides.

Example 6 Silk Gel Dry Massing

Silk gel samples of various 23RGD-ethanol- and ethanol-enhancedformulations were cut into sample cylinders (N=4 of each type) ofapproximately 0.7 cm in height by 0.5 cm in diameter (approximately 500mg). These samples were massed to the nearest 0.1 mg using an APX-60(Denver Instrument, Denver Colo.) balance as per the manufacturer'sinstructions and placed into massed microcentrifuge tubes. After this,the samples were frozen to −80° C. for 24 hours. At the conclusion ofthis time, the samples were placed into a lyophilizer unit for a periodof 96 hours to remove all water content. Following the completion ofthis 96 hour drying, the remaining protein components of the silk gelsamples were massed again and the mass fraction of water in the samplesdetermined.

Gel dry massing showed an increasing percentage of dry mass as RGDcomponent increased in each silk concentration group (FIG. 3). The drymass of 2E was significantly less than 2RL and 2RM (p 0.05) at1.63±0.30%, 3.85±1.23% and 4.03±0.53% respectively (FIG. 3A). The drymasses of 4E, 4RL and 4RM all differed significantly from each other at4.05±0.10%, 4.56±0.12%, and 5.19±0.18% respectively (FIG. 3B). The drymass of 6E was significantly less than both 6RL and 6RM at 5.84±0.15%,6.53±0.28%, and 6.95±0.40% respectively (FIG. 3C).

The gels, regardless of the silk concentration, showed a statisticallysignificant trend toward decreasing percentage of water mass in each gelmaterial as 23RGD component increased as determined by analysis of eachsilk concentration group with ANOVA (FIG. 4, Tukey post hoc, p<0.05).This phenomenon might be explained by the possibility that the 23RGDcauses formation of a different secondary structure within the silkhydrogels and that this structure might be less hydrophilic thannon-23RGD-enhanced material. It is possible that this may be manifestedin a different ratio of β-sheet structure, α-helix structure, andunordered random coil for 23RGD-treated materials than theircounterparts, tending to favor the more hydrophobic β-sheetconformation.

Silk gel dry mass data revealed that increasing concentrations of bothsilk and RGD in the silk gels increased the percentage of dry mass inthese materials, though the increase from RGD was too large to attributesolely to additional peptide mass. This phenomenon might be explained bythe hypothesized structure of the RGDEEG gels mentioned previouslyrelative to PG and EEG gels. It is likely that the large regions ofpoorly-associated β-sheet structure in the RGDEEG gels do a poor job atintegrating water into the structure. The inter-connecting regions ofα-helix structures and unordered random coil are able to entrain water,but do so with less success than in the case of the more homogenous EEGgels. It may also be possible that the hydrophilic RGD sequenceinterfered with the dry massing procedure, causing rapid gain of watermass upon exposure of the samples to atmospheric conditions.

Example 7 Enzymatic Bioresorption

Gels specified were subjected to in vitro digestion by a solutionconsisting of non-specific protease mixture. Briefly, gel samples werecast to generate uniform, cylindrical samples of approximately 1 gramtotal weight (about 1 mL of gel). These samples were digested with aprotease obtained from the bacteria Streptomyces griseus (Sigma catalogNo. P-5147) suspended in phosphate buffered saline at a concentration of1 mg/ml. A ratio of 3 mL of protease solution per 1 ml of initial gelwas used for the purposes of this study. The protease solution was addedto a sealed tube containing the gel and incubated for 24 hours at 37° C.with no mechanical mixing. After 24 hours, the solution was drainedthrough a piece of 316 stainless steel woven wire cloth. This permittedretention of all gel particles greater than 50 μm in diameter (gap sizewas 43 μm by 43 μm), those smaller than that were considered to be“bioresorbed” for the purposes of this assay. After thorough draining ofthe solution, the mass of the gel was measured wet, but devoid of excessentrained moisture. The protease solution was then replaced and thesample incubated a further 24 hours at 37° C. This process was repeateduntil the samples were bioresorbed for a total of four days, changingsolutions and massing each day.

PG samples and EEG samples bioresorbed similarly, differingsignificantly only at D4 where 4P samples retained 62.89±4.26% of theoriginal mass and 4E samples retained 53.27±5.45% (p 0.05) (FIG. 5A). 6Egels incubated in PBS showed no significant mass loss over the course ofthe 4 day incubation (FIG. 5B). EEG silk gels with high concentrationsof fibroin exhibited higher mass retention than lower concentrations atall days. At Day 1 there were significant differences between 2E and allother gel types with 2E, 4E and 6E gels retaining 57.04±10.03%,93.21±9.47%, and 103.98±3.65%, respectively while 6E in PBS retained101.18%±12.01%. At Day 2, there were significant differences againbetween 2E and all other gel types with 2E, 4E and 6E gels retaining36.59±7.07%, 90.60±9.24%, and 103.24±6.38% of the original mass while 6Ein PBS retained 98.28%±12.38%. At Day 3 there were significantdifferences between all gel types in protease, with 2E, 4E and 6E gelsretaining 32.36±10.48%, 67.85±8.82%, and 95.51±8.97% of the originalmass. 6E samples incubated in PBS did not differ from those incubated inprotease, retaining 100.39%±12.73% of the original mass. At Day 4 therewere significant differences between all gel types with 2E, 4E, and 6Egels retaining 28.14±4.75%, 53.27±5.45%, and 81.76%±3.35% of theoriginal mass while 6E in PBS retained 102.45±12.50%. Addition of RGD tosilk gels appeared to slightly decrease the mass retention of thesematerials when subjected to proteolytic bioresorption (FIG. 5C). 4Esamples retained significantly more mass than 4RM and 4RH at Day 2 asthey retained 90.6±9.24%, 74.47±4.55%, and 71.23±6.06% of the initialmasses respectively. There were no further significant differences in 4Esamples relative to 4RM and 4RH samples over the course of thebioresorption assay.

Gel samples treated with 23RGD exhibit a trend toward more rapidbioresorption within the constraints of this particular assay. This wasillustrated at the 4% silk concentration (FIG. 6) and then confirmed ata concentration of 6% silk fibroin in the gel materials (FIG. 7).Significant differences in the bioresorption rates of 23RGD-enhancedsamples recorded by two-way ANOVA using a Bonferroni post test (p<0.05),particularly with 6% silk, reinforced the trend. The unique behaviorattributed to 23RGD-enhanced materials may be due in part to its uniqueprotein structure, as the bioresorption method considers particles belowa size of 50 μm to be bioresorbed, regardless of their stability. It maybe possible for a rich beta sheet structure to exist within 23RGD gelswhich is broken up into small, discrete regions by interfering regionsof α-helix structure and random coil which bioresorb more quickly,creating a plethora of tiny, non-resorbed fragments in solution.

In vitro bioresorption of 4P and 4E samples showed both materials had asimilar resistance to proteolysis (FIG. 5A). This is indicative that thebasic process of ethanol-enhanced gelation is capable of generating agel structure rapidly without sacrificing important material properties.It was also shown that increasing the concentration of silk in EEG gelsfrom 2% to 4% to 6% in 2E, 4E, and 6E respectively, substantiallydecreased sample bioresorption mass loss (FIG. 5B). This may correlateto a more homogeneous, stable and resilient gel structure, or simply toa greater quantity of silk molecules to be cleaved by the proteases inorder to bioresorb the samples. In either case, these data clearlyindicate a potential for tailoring of bioresorption time scale of a silkgel material through alteration of the silk protein content of gels. Itwas also illustrated that a 4 day exposure to PBS did not appreciablyalter the mass of 6E samples, providing a preliminary indication thatEEG samples are not substantially degraded by hydrolysis. This is afurther reinforcement of the stability and bulk integrity of these silkgels as many gel materials suffer from limited resilience in vivo due tohigh susceptibility to hydrolysis. Addition of increasing quantities ofRGD to silk gels was shown to slightly increase the rates ofbioresorption mass loss in comparing 4E, 4RM and 4RH (FIG. 5C). Thisbehavior indicates that there may be some structural differences betweenRGDEEG and EEG gels which cause less mass loss in EEG gels as comparedto RGDEEG in this bioresorption assay. This may relate directly to thepreviously proposed idea that RGDEEG materials consist of many smallregions of robust β-sheet structure loosely bound together by a weakinter-connecting matrix of α-helix and unordered random coil structures.This stands in contrast to EEG materials, which are thought to assemblefrom similar, though less prominent and numerous, precipitates into amore homogeneous structure than RGDEEG gels as a result. Theinter-connecting matrix of the RGDEEG gels is therefore more susceptibleto rapid bioresorption through this proteolytic assay than that of EEGgels. While β-sheet regions may remain intact in RGDEEG gels, bulkmaterial integrity is lost as the inter-connecting network is resorbedas are the residual β-sheet particles due to the sieving method used asa cutoff for degradation product particle size. This is indicative thatit may be possible to use varying levels of RGD in order to furthermanipulate the structure and bioresorption profile of a silk gel.

Example 8 Fourier-Transform Infrared Spectrum Capture

Silk hydrogels, 23RGD-ethanol-enhanced 4% silk, 3:1 and 10:1, were castas described above and subjected to proteolytic bioresorption asdescribed above. Additionally, non-bioresorbed control samples wereobtained for sake of analysis via FTIR in quantities of 0.5 ml each.Using a Bruker Equinox 55 spectrophotometer (Bruker Optics, Inc.,Billerica, Mass.) coupled with a Pike MIRACLE™ germanium crystal (PIKETechnologies, Madison, Wis.), sample absorbance spectra were obtained.Samples were imaged by pressing them upon the crystal via a pressure armuntil single sample scans indicated viable signal from the material thenperforming a 128-scan integration. Resolution was set to 4 cm⁻¹ with a 1cm⁻¹ interval from a range of 4000 cm⁻¹ to 400 cm⁻¹.

Resultant spectra were subjected to analysis via OPUS 4.2 software(Bruker Optics, Inc). A peak-find feature was used to identify peaksbetween 4000 cm⁻¹ and 600 cm⁻¹, with the search criteria being automaticselection of local inflection points of a second-derivative, nine-pointsmoothing function. Program sensitivity was set to 3.5% for all spectrabased upon operator discretion regarding magnitude of peaks identifiedand likely relevance to compound identification and “fingerprinting”.

Each of the samples subjected to FTIR analysis exhibited a spectrum withvery pronounced peaks at the Amide I band (1600-1700 cm⁻¹) (FIG. 8).Additionally, the specific wave numbers of these peaks are consistentbetween the 23RGD-infused silk fibroin hydrogel and other silk gelgroups. All samples exhibit major peaks at ˜1622 cm⁻¹ and a minorpeak/toe region at ˜1700 cm⁻¹, a pattern associated with a high degreeof β-sheet structure within a sample (FIG. 8). There are alsosimilarities across all samples types at the Amide II band with a majorpeak at ˜1514 cm⁻¹.

Use of the EEG process to produce silk gels did not dramatically impactgel secondary structure but did slightly increase the resistance of thegel formulation to proteolytic bioresorption (FIG. 9A). Evaluation ofcharacteristic FTIR spectra of 4P and 4E gels at Day 0 revealed fewdistinguishing characteristics as both formulations exhibited acharacteristic β-sheet peak around 1622 cm-1 and toe region of β-turn at1700 cm-1. Each sample also had additional portions of β-sheet, β-turn,α-helix, and unordered random coil at 1677 cm⁻¹, 1663 cm⁻¹, 1654 cm⁻¹,and 1645 cm⁻¹ respectively with higher relative quantities of α-helixand random coil appearing in 4P than 4E at Day 0. At Day 4, both samplesshowed pronounced decreases in 1677 cm⁻¹ β-sheet, β-turn, α-helix andrandom coil signal, though this 4P exhibited this to a greater extentthan 4E, indicating preferential resorption of these motifs and greaterresistance to this in 4E gels.

Increasing the final silk concentration of EEG gels had little impact oninitial gel secondary structure, though there was a pronounced increasein β-sheet structures at Day 4 with greater silk concentrations (FIG.9B). At Day 0, 2E, 4E, and 6E gels all showed strong signal for 1622cm⁻¹ β-sheet and 1700 cm⁻¹ β-turn strong, with 6E having particularlyprominent peaks in these regions. Each sample also had additionalportions of 1677 cm⁻¹ β-sheet, 1663 cm⁻¹ β-turn, α-helix, and unorderedrandom coil. At Day 4 all gels showed decreases in 1677 cm-1 β-sheet,1663 cm⁻¹ β-turn, α-helix and random coil peaks relative to 1622 cm⁻¹β-sheet and β-turn peaks with this behavior being more marked in 4E and6E than 2E. The Day 4 6E sample also showed a more stable β-sheetstructure indicated by a peak shift to lower wave number at ˜1620 cm⁻¹.

Pronounced differences in the 23RGD-ethanol-enhanced andethanol-enhanced spectra only became evident after a four-day period ofbioresorption in protease. The day 4 samples exhibited differencesprimarily in the order of magnitude of certain secondary structuremodalities seen through slight differences in FTIR Amide I band shape.At day 4, the 23RGD-ethanol-enhanced samples exhibit higher levels ofβ-turn structure evidenced by far more pronounced and distinct peaks at˜1700 cm⁻¹ while also showing considerably lower levels of α-helixstructure (1654 cm⁻¹) and unordered random coil (1645 cm⁾ structures.For example, FTIR spectra from 4E, 4RM and 4RH all show similarstructures featuring 1622 cm⁻¹ β-sheet and 1700 cm⁻¹ β-turn prominentlywith indications of 1677 cm⁻¹ β-sheet, 1663 cm⁻¹ β-turn, α-helix, andunordered random coil secondary structures (FIG. 9C). At Day 4, 4RM and4RH both show a less pronounced 1677 cm⁻¹ β-sheet, 1663 cm-1 β-turn,α-helix, and random coil component than the 4E sample with 4RH alsoshowing a more stable β-sheet structure, indicated by a peak shift tolower wave number at −1620 cm⁻¹. Additionally, a peak shift occurred inboth the 10:1 23RGD-ethanol-enhanced and ethanol-enhanced samples in theβ-strand peak at 1622 cm⁻¹, indicative of increased β-sheet stability.Considered as a whole, the collective peak shifts and peak magnitudesobserved in the spectra at day 4 compared to day 0, all gel typesexperienced substantial strengthening of β-sheet component, likely dueto removal of less-stable α-helix and random coil. These effects weremost pronounced in 23RGD-enhanced gel materials, likely due to intrinsicdifferences in the initial organization of the structural network of thegel materials.

FTIR analysis and comparison of PG, EEG and RGDEEG showed strongbehavioral similarities across all gel groups. Each material exhibitedβ-sheet-dominated secondary protein structures, featuring elements ofα-helical and random coil structures and each resorbed in such a fashionthat the quantities of β-sheet-rich structure increased relative toα-helical and random coil structures. The selective bioresorption ofα-helical and random coil structures indicates that they are likelyfavorably degraded by proteolysis relative to β-sheet structures, thusthe bioresorption profile of a gel might be influenced by altering thebalance between β-sheet motifs and the combination of α-helical andrandom coil structures. An evaluation of ethanol as an accelerantrevealed a minimal effect on silk gel structure at Day 0 as both 4P and4E had high β-sheet contents with α-helical and random coil structures(FIG. 9A). At Day 4 though, there was a slightly greater relativeβ-sheet content in 4E than 4P samples. This may be caused by structuraldifferences in 4E and 4P formulations that were imperceptible at Day 0by ATR-FTIR, possibly in the uniformity and homogeneity of the silkgels. It is possible that the same differences hypothesized between EEGand RGDEEG gels derived from their different extents ofprecipitate/nucleation point formation in early-phase gelation causesdifferences between PG and EEG materials as well. As PG samples are notaccelerated, it is likely that very few nucleation points will formquickly and as a result, the gelation process occurs in a very slow buthomogeneous fashion, allowing for an optimal stearic packing of silkmolecules throughout the solution volume. This results in a consistentprotein structure throughout the final gel volume, corresponding to goodbulk material integrity. This would contrast with EEG gels, as thepreviously postulated nucleation phenomenon associated with RGDEEGmaterials likely occurs with EEG materials as well, though in a lessprominent fashion. This results in a non-uniform distribution of highlyorganized regions of β-sheet held together by α-helical and random coilstructures in the EEG materials relative to the PG materials, withα-helical and random coil degraded more rapidly than β-sheet. This is inkeeping with previous studies which have shown that more poorly packedβ-sheet structures and α-helix structures are more susceptible todegradation. Increasing silk concentration in EEG gels from 2E to 4E to6E revealed the most prominent β-sheet structures in 6E at both Day 0and Day 4 while 2E had considerably more α-helix and random coil at bothdays than 2E and 4E (FIG. 9B). This would seem to indicate that diluteconcentrations of silk in the final hydrogel result in a less denselypacked secondary structure, possibly due to stearic freedom within thegel volume relative to 4% and 6% states. This indicates that silkconcentration may be used to manipulate the secondary structure of silkgel to influence bioresorption. A study of the effect of increasing RGDconcentration indicated that while gels were virtually identical at Day0, the α-helix structure and unordered random coil in 4RM and 4RH gelswere less resilient to bioresorption than in 4E as seen at Day 4 (FIG.9C). This might also be explained by inhomogeneities within the 4RM and4RH gels relative to 4E as mentioned previously. This may beparticularly likely in light of the formation of precipitates observedin RGDEEG samples. This data may be indicative that RGD or a similarpeptide could be used to further tailor the nature of the bioresorptionprofile of silk gels.

These results indicate that silk gels produced through PG, EEG, andRGDEEG result from a two-phase assembly process consisting of nucleationand aggregation. Silk gels contain predominantly β-sheet structure whichis more resistant to in vitro bioresorption than α-helix and randomcoil. EEG gels form more quickly than PG, likely due to a more rapidprecipitation and nucleation event mediated by the effects of ethanol onthe solution solvent phase. EEG gels form a non-homogeneous structurelikely consisting of localized, initially-precipitated β-sheet regionsinter-connected by α-helix and random coil assembled subsequently.RGDEEG gels form a non-homogeneous structure likely consisting oflocalized, initially-precipitated β-sheet regions inter-connected byα-helix and random coil assembled subsequently. RGDEEG gels reachcompletion more slowly than EEG gels due to stearic RGD-mediatedinterference encountered in gel assembly following nucleation. RGDEEGgels are less homogeneous than EEG gels due to these difficultiesassociated with late-phase assembly.

Example 9 Injectable Gel Processing

Silk hydrogels were prepared as described above in Examples 1-4. Gelswere then comminuted by grinding the silk gel to a paste using astainless steel spatula. Gel formulations including PBS were massed withan SI-215 balance (Denver Instrument, Denver Colo.) and the correctvolume percentage of PBS (Invitrogen Corporation, Carlsbad, Calif.) wasblended in with the assumption that both the gel and PBS had a densityof 1 g/ml. Silk hydrogels to be used for in vivo assessment weresubjected to vigorous mechanical pulverization by means of a stainlesssteel stir rod. When specified as containing a saline component, gelswere blended with saline at volumetric ratios based upon the originalvolume of gel (i.e., prior to mechanical disruption) followingpulverizing by the stainless steel bar. This addition of phosphatebuffered saline serves to regulate tonicity of the gel as well asimprove injectability. Following this initial pulverizing, the gel wasfurther disrupted by means of repeated injection through a 26-gaugeneedle in order to decrease overall particle size within the gel andimprove injectability characteristics. In some samples, gel was furtherdisrupted by means of repeated injection first through an 18 g needlerepeatedly until the gel flowed readily, and then the material was thencycled in like fashion through a 23 g needle and 26 g needle.

Example 10 In Vivo Investigation of Silk Hydrogel in Rodent Models

Samples of silk gel which had been processed for implantation orinjection in vivo as described in Example 9 were double-bagged withappropriate sterilization bags for gamma irradiation and sterilized byexposure to a dose of 25 kGy of gamma radiation.

In one trial silk hydrogel samples, both 23RGD-enhanced and native wereimplanted subcutaneously in male Lewis rats having an average weight of400 g. This was done according to protocol#86-04 on file with NewEngland Medical Center's Department of Laboratory Animal Medicine (DLAM)and approved by the Institutional Animal Care and Use Committee (IACUC).On the day of surgery, animals were anesthetized via a ketamine/xylazinesolution injected IM in the animals' hind legs. Following administrationof anesthesia, the skin of the rats was shaved closely and swabbed withalcohol, allowed to dry, swabbed with BETADINE® microbicide (PurduePharma, Stamford, Conn.) then draped with sterile towels. In the case ofimplanted devices, two dorsal midline incisions were made directly overthe spine, the first 0.5 cm below the shoulders and the second 2.5 cmabove the pelvic crest, each 1 cm long each. The incisions were expandedinto 1 cm deep pockets using a blunt dissection technique beneath thepanniculus carnosus at each side yielding 4 potential implant sites.Implants, 3 per animal; each 1 cm×1 cm×0.3 cm in size were inserted intothe pockets without fixation with the final site undergoing the samedissection but replacing the implant with 0.5 mL of sterile salinesolution. The skin was closed with interrupted absorbable sutures.Depending on study, samples were harvested at 7 days, 14 days, 28 days,and/or 57 days after implantation surgery. Gross observations werecollected semi-weekly regarding implant site appearance. After sampleharvest, gross observations of the implants were conducted and sampleswere processed for histological evaluation. Analysis of histology slideswas provided by a trained veterinary pathologist.

Sections were scored for presence (0=none, 1=present) of implantmineralization, cyst formation, fibrosis, sebaceous cell hyperplasia,and focal follicular atrophy. Additionally, the density of inflammatoryresponse (0=none . . . 5=extensive) and extent epidermal hyperplasia(0=none . . . 3=extensive) were graded. These data were reported aspercentages of the highest score possible for the group of slides.Sections were also examined for presence of any particularcharacteristic cell types including lymphocytes, neutrophils,eosinophils, mononuclear giant cells, macrophages, and fibroblasts.Additional commentary relevant to the host response was included at thediscretion of the reviewing pathologist. Prism 4.03 (GraphPad SoftwareInc., San Diego, Calif.) was used to perform analysis of variance(ANOVA) with a significance threshold set at p≦0.05. One-way ANOVA wasused to compare differences average extrusion forces for comminutedgels. For all tests, Tukey's post-hoc test was also performed formultiple comparisons.

Table 2 lists the formulations of silk gel, both 23RGD-ethanol-enhancedand ethanol-enhanced developed and assessed intradermally in a ratmodel. Silk gels explanted from rats at Day 7 were visibly well-definedand easily identifiable with no gross indications of edema, erythema, ortransdermal elimination of material. It was not possible todifferentiate sites of PBS control implantation from surrounding tissue.H&E sections of 4% silk fibroin hydrogels formed by passive gelation(4P), 4% silk fibroin hydrogels formed by ethanol-enhanced gelation (4E)and 6% silk fibroin hydrogels formed by ethanol-enhanced gelation (6E)all appeared similar, with mild inflammation in all cases characterizedby lymphocytes, macrophages, some neutrophils and fibroblasts (FIG. 10).Cellular infiltration was observed in all sample types with completepenetration in 4P and peripheral ingrowth to a depth of about 100 μm inboth EEG gels with no evidence of cyst formation observed. In all gels,early bioresorption was indicated by implant edge erosion with residualimplant material remaining localized into large lakes. Host integrationof implanted gel had progressed in Day 28 samples of 4E and 6E evidencedby greater cellular ingrowth into the material with complete implantpenetration in 4E samples and robust peripheral ingrowth in 6E samples.The cellular response at this time point was characterized byfibroblasts, lymphocytes and macrophages with the addition of a fewmulti-nucleated giant cells.

TABLE 2 Silk Hydrogel Formulations Group Silk Saline Name ConcentrationEnhancer Component 4E10 4% 90% Ethanol 10% 4R10 90% Ethanol, 1:1 23RGD4RH10 90% Ethanol, 3:1 23RGD 4E25 90% Ethanol 25% 4R25 90% Ethanol, 1:123RGD 4RH25 90% Ethanol, 3:1 23RGD 6E10 6% 90% Ethanol 10% 6R10 90%Ethanol, 1:1 23RGD 6E25 90% Ethanol 25% 6R25 90% Ethanol, 1:1 23RGD6RH25 90% Ethanol, 3:1 23RGD

Day 57 samples of 4E and 6E showed continued host bioresorption of thegel material as there was little residual 4E and while 6E remainedvisible in large, intact lakes, the gel had been completely penetratedwith host tissue. The host response to 4E had dramatically decreased incellularity between Day 28 and Day 57 with very little evidence ofhypercellularity at Day 57 with some scattered macrophages andfibroblasts around the implant site. The pathology of the host responseof 6E was similar to the Day 28 response to 4E, with fibroblasts as thepredominant cell type and scattered lymphocytes, macrophages andmulti-nucleated giant cells. This was viewed as a low-grade, persistent,fibrotic-type inflammatory response to the material.

Samples of 23RGD-enhanced gel exhibited a less robust inflammatoryresponse at the 14 day time point in comparison to non-23RGD-enhancedgel (FIG. 11). This is observed through an appreciable decrease inhyper-cellularity proximal to the gel implant and an accompanyingdecrease in the fragmentation of the implant material. It is possiblethat this improvement in implant integrity is due to a less robustforeign body response by the host animal and it may also be evidencethat there is less mechanical contraction of the implant site, acommonly observed phenomenon with biomaterials including the “RGD”motif. These effects indicate that 23RGD-enhancement of silk gels leadsto a more biocompatible material with better implant outcomes.

In a second trial, intradermally-injected samples of silk hydrogel, bothethanol enhanced and 23RGD-ethanol enhanced and relevant controlmaterials were investigated using male Hartley guinea pigs. This wasdone according to protocol#29-05 on file with New England MedicalCenter's Department of Laboratory Animal Medicine (DLAM) and approved bythe Institutional Animal Care and Use Committee (IACUC). Briefly, maleHartley guinea pigs weighing 300-350 g were anesthetized via aketamine/xylazine cocktail injected intramuscularly into the animals'hind legs. The dorsal skin of the guinea pigs was then shaved closelyand swabbed with alcohol, allowed to dry, swabbed with BETADINE®microbicide or Chloraprep (Enturia, Inc., Leawood, Kans.), then drapedwith sterile towels. A 50 μL volume of the desired material was injectedthrough a 26 g needle at six different sites along the left side of theanimal's back. Further injections of an appropriate silk gel controlwere made at the six contralateral sites. Explanation of the silk gelswas performed at 28 days after implantation. Gross observations werecollected semi-weekly regarding implant site appearance. After sampleharvest, gross observations of the implants were conducted and sampleswere processed for histological evaluation. Analysis of histology slideswas provided by a trained veterinary pathologist. Scoring andstatistical analysis was performed as described above.

Table 3 lists the formulations of silk gel, both 23RGD-ethanol-enhancedand ethanol-enhanced developed and assessed intradermally in a guineapig model in a twenty-eight day screen. Although no statisticallysignificant differences were identified, the data for both grossobservations and histology (Tables 4 and 5) indicate a general trendsupporting the previous data that 23RGD-enhancement of gel improvesmaterial biocompatibility. Among sites implanted with silk gel, grossoutcomes varied. Ulceration and hair loss rates were lower in groupswith 25% PBS compared to 10% saline, 6% silk compared to 4% silk andRGDEEG casting as compared to just EEG casting (Table 4). Site rednessrates followed a similar pattern with the exception that RGDEEG samplesinduced more site redness than EEG samples. All silk gels showedevidence of epidermal cyst formation, fibrosis, epidermal hyperplasiaand pronounced inflammation with traces of follicular atrophy in all EEGsamples. Sebaceous cell hyperplasia was present to a limited extent inall formulations with the exception of 6% silk, 10% saline, 1:1 23RGD(Table 5). This is particularly evident in the case of silk gels of 4%silk with 25% saline added and either enhanced with an ethanol-basedenhancer or an 23RGD-ethanol-based enhancer, and more specifically, inthe case of site ulcerations (Table 5). This material indicated strongimprovements with increasing 23RGD concentration in the number of sitesulcerating throughout the course of the trial. These results areindicative that use of 23RGD in conjunction with an ethanol enhancerprovides an improved outcome when compared to an ethanol enhancer alone.

TABLE 3 Silk Hydrogel Formulations Group Silk Saline Name ConcentrationEnhancer Component 4E10 4% 90% Ethanol 10% 4R10 90% Ethanol, 1:1 23RGD4E25 90% Ethanol 25% 4R25 90% Ethanol, 1:1 23RGD 4RH25 90% Ethanol, 3:123RGD 6E10 6% 90% Ethanol 10% 6R10 90% Ethanol, 1:1 23RGD 6E25 90%Ethanol 25% 6R25 90% Ethanol, 1:1 23RGD

TABLE 4 Gross Evaluation of Guinea Pigs Group Site Hair Name RednessLoss Palpability Ulceration 4E10 38% 58% 65% 33% 4R10 57% 49% 67% 33%4E25 28% 34% 49% 28% 4R25 44% 34% 64% 17% 4RH25 50% 23% 66%  6% 6E10 63%52% 68% 33% 6R10 78% 51% 68% 22% 6E25 33% 31% 69% 11% 6R25 56% 30% 68%13% HYLAFORM ™  6% 12% 63%  0% ZYPLAST ™ 17% 10% 52%  0%

TABLE 5 Histological Evaluation of Guinea Pigs Epidermal Cyst EpidermalFollicular Sebaceous Group Name Formation Fibrosis InflammationHyperplasia Atrophy Hyperplasia 4E10 22% 100% 70% 59% 11%  22% 4R10 74%100% 62% 67% 0% 14% 4E25 50% 100% 69% 67% 13%  13% 4R25 29% 100% 39% 62%0% 14% 4RH25 14% 100% 64% 50% 0% 43% 6E10 44% 100% 70% 56% 11%  33% 6R1025% 100% 63% 38% 0%  0% 6E25 30% 100% 60% 40% 10%  20% 6R25 29% 100% 64%33% 0% 14% HYLAFORM ™  0%  0%  3%  6% 0%  0% ZYPLAST ™  0%  25% 28% 31%0%  0%

A third trial also used male Hartley guinea pigs to investigateintradermally injected samples of silk hydrogel as described above,comparing samples of 4% and 6% silk, 25% saline 3:1 23RGD-ethanolenhanced silk gels with a collagen-based control material, ZYPLAST™(Allergan Inc., Irvine Calif.) and HYLAFORM™ (Allergan Inc., IrvineCalif.). Explanation of the silk gels was performed at 92 days afterimplantation. Gross observations were collected semi-weekly regardingimplant site appearance. After sample harvest, gross observations of theimplants were conducted and samples were processed for histologicalevaluation. During the course of the 92 day trial, none of the 24implant sites, either 23RGD-ethanol-enhanced hydrogel or ZYPLAST™,ulcerated. Histology revealed that 75% of all ZYPLAST™ sites hadresidual material as did 75% of all 23RGD-ethanol-enhanced silk gelsites (both 4% and 6%). Both materials exhibited very similar chronicphase cellular responses, as the sites were characterized by a mildfibrotic reaction with abundant deposition of collagen in and around theimplant site (FIG. 12). The collagen appears less ordered than does thatin the surrounding dermal reticulum based upon the color density whenviewed with Trichrome staining and also when viewed under polarizedlight. Silk gel sites had similar palpability scores to both controlmaterials but exhibited higher rates of site redness, hair loss andulceration than did ZYPLAST™ and HYLAFORM™. These results not onlyreinforce that 23RGD-ethanol-enhanced silk gel is biocompatible, butalso indicate that it is comparable to collagen biomaterials in terms ofits persistence and long-term behavior in vivo.

ZYPLAST™ exhibited no epidermal cysts, follicular atrophy, or sebaceouscell hyperplasia, though it did show small levels of fibrosis,inflammation and epidermal hyperplasia. Examination of histologicalsections showed residual silk gel material which stained in a mildlyeosinophilic fashion and appeared as large lakes of material at acentral location with smaller masses of material distributed more widelythroughout the reticular dermis (FIG. 13). These smaller masses weretypically surrounded by fibroblasts and macrophages with occasionalmulti-nucleated giant cells present. Eosinophils were located proximalto these smaller masses of implant as well. In general, host response tothe silk fibroin gels was characterized as mildly fibrotic and includedpopulations of fibroblasts, lymphocytes, macrophages, multi-nucleatedgiant cells and eosinophils. Little difference was evident between silkgel types except in terms of the extent of eosinophilia. Largereosinophil populations were observed for 6% as compared to 4% silk gelsand were also observed to increase with RGD concentration in the silkgel samples in both 4% and 6% groups. ZYPLAST™ exhibited strongeosinophilic staining and was distributed as large lakes in thereticular dermis with smaller masses throughout the area.Hypercellularity near the injection site was lessened in ZYPLAST™samples when compared to silk gel. Fibroblasts, lymphocytes,macrophages, multi-nucleated giant cells and eosinophils were presentwith less tendency to localize at the implant periphery. HYLAFORM™samples examined showed many very small masses of material throughoutthe reticular dermis. HYLAFORM™ exhibited no epidermal cysts, fibrosis,follicular atrophy, or sebaceous cell hyperplasia with extremely limitedinstances of inflammation and epidermal hyperplasia. There was noobservable hypercellularity near the implanted material or otherevidence of inflammation at the implant sites.

At day 92, histological evaluation of 4% silk fibroin hydrogel, 3:123RGD, 25% saline (4RH25) samples and ZYPLAST™ samples showed similarmaterial persistence and host response (FIG. 14). Very little implantmaterial remained visible in the dermis of the animals with nohypercellularity present at this time point, evidence of hyperplasia orcellular inflammation. The eosinophils found at day 28 in the ZYPLAST™and silk gel samples were not observed at day 92. Of particularinterest, 4RH25 also exhibited residual disruption to the reticulardermis in the form of an irregular collagen pattern near the implantmaterial. The disorganization of the collagen was seen as a region ofstained collagen seen to be devoid of the typical cross-hatch pattern ofnormal reticular dermis (FIG. 14C). This disorganization was confirmedwhen viewing the histological sections under polarized light with thedisorganized collagen appearing as an interruption in the birefringenceassociated with the surrounding reticular dermis (FIG. 14D).

Example 11 Enhanced Injectable Gel Formulation

Silk hydrogels were prepared as described above in Examples 1-4. Onceprocessed, the gels were sized into coarse or fine particles using asieving step (Table 6). Gel materials were pressed through a 316SSstainless steel wire cloth sieve with a stainless steel spatula and intoclean polystyrene Petri dishes. Sieves with gap sizes of 711 μm×711 μm,295 μm×295 μm, 104 μm×104 μm and 74 μm×74 μm were used. After passingthrough the 74 μm×74 μm gap sieve, the material was considered processedto a “coarse” state. Samples to be processed to a “fine” state werefurther forced through a 43 μm×43 μm sieve in the same fashion. Thissieving was conducted four separate times for each sample type, eachsieving using an approximate quantity of 0.5 mL of gel material.

TABLE 6 Particle sizing Nominal Silk 23RGD Molar Ratio Group MassPercentage with Silk Fineness Name 2% 1:1 Fine 2RF 4% 0 4F 1:1 Coarse4RC Fine 4RF 3:1 RHRF 10:1  4VHRF 8% 1:1 Coarse 8RC Fine 8RF

Samples of silk gel material (N=4 of each type) were evaluated underlight microscopy. Briefly, a 100 mg portion of silk gel or controldevice was massed using an SI-215 Summit series balance. This materialwas loaded into the open back end of a 3 mL syringe using a stainlesssteel spatula. The plunger was replaced in the syringe, an 18 g needlewas attached to the end of the syringe and approximately 900 μL ofultra-pure water was drawn up. This mixture of water and silk gel wasmixed through gentle shaking. After mixing to suspend evenly, a sampleof approximately 30 μL of dilute silk gel was placed on a 75 mm×25 mmsingle frosted, pre-cleaned micro slide (Fisher Scientific Co., Waltham,Mass.) and covered with a 22 mm×40 mm premium cover glass (Corning Inc.,Corning, N.Y.). This sample slide was then be imaged with a microscope.Sample slides were imaged using a System Microscope Model BX41 (Olympus,Melville, N.Y.) in conjunction with a Microscope PC MACROFIRE™ ModelS99831 Camera (Optronics, Goleta, Calif.) and PICTUREFRAME™ 2.1 software(Optronics, Goleta, Calif.). Briefly, slides were scanned for clearlyseparated gel particles using the 4× objective lens and locationsdetermined for a series of 3 representative images of the sample slide.Each of these locations was imaged after first switching the microscopeobjective lens to 10×. Micrograph image files were subjected to analysiswith IMAGE-PRO® Plus 5.1 software (Media Cybernetics, Inc., SilverSpring, Md.). Image files were checked for particle size distribution,average particle size, average aspect ratio, maximum particle size,minimum particle size and standard particle size deviation. Acompilation of the data is presented in Table 7.

TABLE 7 Particle Comminution Data Group Min to Max Object Mean ObjectName Area (μm²) Area (μm²) 2RF 5.33 to 1.32 × 10⁴ 52.43 ± 261.82 4F 5.33to 8.07 × 10³ 27.82 ± 129.34 4RC 5.33 to 8.52 × 10³ 38.41 ± 196.67 4RF5.33 to 5.29 × 10³ 34.12 ± 135.31 4HRF 5.33 to 7.51 × 10³ 40.62 ± 166.614VHRF 5.33 to 3.14 × 10³  35.4 ± 105.43 8RC 5.33 to 8.04 × 10³ 46.57 ±225.43 8RF 5.33 to 2.85 × 10³ 35.26 ± 129.63 ZYPLAST ™ 5.33 to 1.95 ×10³ 22.08 ± 41.71 

Examination of the particles under light microscopy revealed someclumped gel particles which were removed from particle sizing datamanually. Particle sizes ranged from 5.3 to 1.3×10⁴ μm², comparable inrange to commercially available ZYPLAST™ which ranged from 5.3 to1.95×10³ μm². The data also revealed mean particle sizes ranging from27.8 μm² to 52.4 μm², again, comparable to ZYPLAST™ with a mean particlesize of 22.1 μm². These data illustrate that silk gel may besuccessfully comminuted to small and functionally useful particle sizesin a fashion similar to presently utilized injectable gel materials. Thebasic forced-sieving method could easily be replaced with moresophisticated, reproducible methods for purposes of scale-up.

After comminution and blending, samples of silk gel emulsions weresubjected to extrusion force testing. Gel materials prepared asdescribed in Examples 1-4 were blended with appropriate ratios of salinein order to evaluate injection (extrusion) force profiles relative to acontrol material, ZYPLAST™ (Table 8). This was accomplished by massing 5g of gel material in a large weighing boat using an SI-215 balance(Denver Instrument, Denver, Colo.). An appropriate quantity of salinewill be added to constitute the correct volume percentage making theassumption that both the gel material and saline have a density of 1g/mL. This material was then blended to an even consistency using astainless steel spatula and loaded into the back end of a 10 mL syringewith an 18 g needle attached for subsequent use.

TABLE 8 Silk Gel Injection Force Profile Generation Nominal Silk 23RGDMolar Ratio Saline Group Mass with Silk Fineness Content Name 2% 1:1Fine 25% 2RF25 4% 0 25% 4F25 1:1 Coarse 25% 4RC25 Fine  0% 4RFO 25%4RF25 50% 4RF50 3:1 25% 4HRF25 10:1  25% 4VHRF25 8% Fine 25% 6RF25

These samples were tested using an Instron 8511 (Instron Corp., Canton,Mass.) in conjunction with Series IX software and a custom-designedaluminum frame attached to a 100 N load cell (FIG. 15). For the materialtesting, 1 mL of the sample material of interest was loaded into a 1 mLgas-tight glass syringe. The sample syringe was mounted in thecustom-designed aluminum frame mounted on the Instron unit and thematerial extruded. The sample was then checked for the force required toextrude the gel at each of 3 strain rates, 10 mm/minute, 50 mm/minute,and 200 mm/minute with total actuator displacement set at 7 mm. A seriesof four tests were run on each material type at each piston displacementrate. Load-displacement data was collected at a frequency of 100 Hz andare presented as the mean±the standard deviation of the 4 averageextrusion forces experienced of each gel type at each strain rate. Theaverage extrusion force was defined as the average load measured in theplateau region of the load-displacement curve resultant from eachextrusion test. The data were reported as the average amount of forcerequired for extrusion of the sample material and are compiled in Table9 and FIG. 16.

TABLE 9 Average Force (N) to Extrude Silk Gel from 30 g Needle PlungerDisplacement Rate 10 mm/min 50 mm/min 200 mm/min Group Name Ave StdevAve Stdev Ave Stdev 2RF25 0.6 0.0 2.9 0.6 7.3 0.7 4RF25 3.7 2.0 4.5 1.322.4 6.7 4RD25 7.1 3.7 6.7 0.5 25.1 3.9 4RF0 9.5 1.0 28.5 3.1 66.2 10.04RF26 3.2 0.9 7.4 0.6 30.4 5.0 4RF50 1.2 0.2 2.7 0.1 10.1 0.3 4HRF25 2.20.4 8.9 1.0 22.0 0.6 4VHRF25 2.8 1.6 5.2 1.4 14.6 2.1 8RF25 3.6 0.7 10.11.3 29.2 2.4 ZYPLAST ™ 1.6 0.5 18.7 0.7 29.1 1.4

A comparison between milling techniques revealed that there were nosignificant differences between 4RC25 and 4RF25, having averageextrusion forces of 7.1±3.7N and 3.2±0.9N at 10 mm/min, 6.7±0.5N and7.4±0.6N at 50 mm/min, and 25.1±3.9N and 30.4±5.0N at 200 mm/minrespectively (Table 6, FIG. 16A). Both of these formulations differedsignificantly (p 0.05) from ZYPLAST™ at strain rates of 10 and 50mm/min, which had extrusion forces of 1.6±0.5 N, 18.7±0.7 N, and29.1±1.4 N at 10, 50, and 200 mm/min strain rates.

Data regarding the extrudability of silk gel formulations clearlyillustrated that the addition of saline as a carrier fluid to thecomminuted silk particles offers an improved degree of extrudability,substantially reducing the force necessary to extrude silk gel at allstrain rates. Adding increasing concentrations of saline to thecomminuted silk gels significantly decreased the extrusion forcerequired for silk gels at each strain rate, with gels again exhibitingshear-thickening behavior (Table 9, FIG. 16B). At all strain rates, 4RF0required significantly more force to extrude than 4RF25, which in turnrequired significantly more than 4RF50. At a strain rate of 10 mm/min,4R0, 4R10, and 4R25 showed a significant decrease (p≦0.05) in extrusionforce with increasing PBS concentration, having average forces of9.5±3.1 N, 6.1±0.5 N, and 4.7±0.7 N respectively (Table 9). At 50mm/min, these relationships were more pronounced with average extrusionforces of 14.0±0.9 N, 5.4±0.7 N, and 3.9±0.2 N respectively and alldiffered significantly (Table 6, FIG. 16). At 200 mm/min, the trendremained as average extrusion forces were 26.4±4.5 N, 10.6±1.6 N, and6.4±0.5 N respectively with 0% PBS differing significantly from theother two groups. Samples of 6R25 had an average extrusion force of29.3±4.8 N at 10 mm/min, significantly higher than 4R25 (Table 9). At 50mm/min and 200 mm/min, the force to extrude the 6R25 was greater than 80N, causing the test to abort in order prevent damage to the load cell.

The data also illustrate that use of very low concentrations of silk mayimprove the extrudability of gel relative to higher concentrations as inthe case of 2RF25 as compared to 4RF25 and 8RF25. Increasing theconcentration of silk in the comminuted silk gels increased theextrusion force required for silk gels at each strain rate, withsignificant increases between 2RF25 and both 4RF25 and 8RF25 at 10mm/min and 200 mm/min (Table 9, FIG. 16C). All groups differedsignificantly at the 50 mm/min strain rate and gels continued to exhibitshear-thickening behavior, seen in the increased extrusion forcesassociated with increased strain rates. At 10 mm/min 2RF25 and 8RF25required 0.6±0.0 N and 3.6±0.7 N respectively, at 50 mm/min theyrequired 2.9±0.6 N and 10.1±1.3 N, and at 200 mm/min 7.3±0.6 N and29.2±2.4 N.

The data further indicated that use of 23RGD to enhance the silk gelmaterial did not appreciably impact the force necessary to extrude silkgel formulations. Adding increasing concentrations of RGD did not have aconsistent effect upon the extrusion force necessary for the gelmaterials (Table 9, FIG. 16D). At a 10 mm/min strain rate there were nosignificant differences between 4F25 at 3.7±2.0 N, 4R25, 4HR25 at2.2±0.4 N, and 4VHR25 at 2.8±1.6 N. At a 50 mm/min strain rate 4HR25 wassignificantly higher than all other extrusion forces at 8.9±1.0 N ascompared to 4F25 at 4.5±1.3 N, 4R25, and 4VHR25 at 5.2±1.4N. At a 200mm/min strain rate 4HR25 at 22.0±0.6 N was significantly higher thanonly 4VHR25 at 14.6±2.1 N as compared to 4F25 at 22.4±6.7 N and 4R25.

Lastly, the data showed that silk gels blended with saline had verysimilar extrudability to ZYPLAST™, a material already proven to bereadily handled as an injectable material. Based upon this data it isbelieved that through careful manipulation of the carrier speciesassociated with the silk gel, modulation of silk concentration, andcontrol of particle size, silk gel materials may be made to behave as areadily injectable material.

These results indicate that silk gels may be comminuted to a particlerange of about 25-50 μm² in cross-sectional area. Silk gels may becomminuted to a size similar to ZYPLAST™. Silk gel particle size can bedecreased by increasing silk concentration or by changing the method ofcomminution. Increasing concentrations of RGD did not develop a cleartrend in silk particle size. Silk gels may be extruded at a relevantstrain rate of 50 mm/min at a force comparable to or less than ZYPLAST™.Silk gel extrusion force may be decreased by adding increased quantitiesof saline carrier or decreased concentrations of silk in the originalgel. Changes of comminution method attempted in this study did notsubstantially affect the amount of force necessary for silk extrusion.Increasing concentrations of RGD did not develop a clear trend in silkgel extrusion force.

Example 12 Silk Gel Precipitates

The silk gel precipitate materials outlined in Table 10 were generatedfor analysis. Silk solution of the specified concentration was generatedusing the stock solution of 8% (w/v) aqueous silk and diluting withpurified water (Milli-Q purified). 23RGD/ethanol accelerant was preparedby generating a solution of ethanol and purified water, then dissolvingthe specified 23RGD quantity by vortexing. Silk precipitates weregenerated by directly adding the specified volume of accelerant solutionto that of silk solution in 50 mL centrifuge tubes, shaking once to mixand allowing the mixture to stand for 5 additional seconds before addingabout 45 mL purified water to halt the gelation process. This materialstood for 24 hours under ambient conditions and was then strainedthrough stainless steel cloth with 150 μm×150 μm pores to recoverprecipitates. These precipitates were rinsed twice for 24 hours in 50 mLof purified (Milli-Q) water at room conditions, strained a final timeand used for evaluation.

TABLE 10 Silk Gel Precipitate Types Generated Initial Silk Solution23RGD/ethanol Accelerant Final Precipitate Silk Silk Ethanol 23RGDAccelerant Final Silk RGD:Silk Concentration Solution ConcentrationConcentration Solution Concentration Molar Group Name (mg/mL) Volume(mL) (%) (mg/mL) Volume (mL) (mg/mL) Ratio BASE 80 1 90 2.45 1 40 5.0RHI 80 1 90 4.90 1 40 10.0 RVLO 80 1 90 0.49 1 40 1.0 RLO 80 1 90 1.47 140 3.0 SCLO 80 1 90 2.45 1 30 6.7 SCVLO 80 1 90 2.45 1 20 10.0 ECLO 80 180 2.45 1 40 5.0 ECVLO 80 1 70 2.45 1 40 5.0 AVHI 80 0.67 90 2.45 1.3327 10.0 AVLO 80 1.33 90 2.45 0.67 53 2.5

Samples of gel were examined under low-vacuum conditions (˜1 Torr) on aQuanta 200 (FEI Co., Hillsboro, Oreg.) environmental scanning electronmicroscope with images collected at magnifications of 200×.Representative images were taken to illustrate surface topographycharacteristics of silk precipitate samples (FIG. 17). All silkprecipitate types appeared similar under ESEM analysis. Each sampleexhibited a mixture of both granular and filamentous regions withoccasional appearance of large, contiguous masses of smooth material.

Example 13 Silk Gel Precipitate Massing

Silk precipitate samples, as described in Example 12, were isolatedafter rinsing by straining through stainless steel wire cloth with apore size of 104 μm×104 μm and gently blotted with a clean, lint-freewipe. Samples were massed to the nearest 0.01 mg using an S-215 balance(Denver Instrument, Denver, Colo.). These samples were frozen to −80° C.for 24 hours and placed into a Labconco lyophilizer unit (LabconcoCorp., Kansas City, Mo.) for 96 hours to remove all water content. Theprecipitate residual solids were massed again and the dry mass fractionin the samples determined. One-Way analysis of variance (ANOVA) was usedto test for significant differences caused by changing silkconcentration, 23RGD concentration and accelerant volume. A Student'st-test was used to test the significance of differences resulting fromaltered ethanol concentrations.

Increasing silk fibroin concentration increased precipitate dry masswith Increasing the percentage of ethanol in the accelerant solutionalso increased dry mass of the precipitates with ECVLO produced onlytrace quantities of precipitate (visible, but not recoverable inmeasurable quantities).

Increasing accelerant volume significantly increased precipitate drymass as AVHI was significantly greater than both AVLO and BASE (p 0.05,FIG. 18A). For example, AVHI (18.02±3.9 mg) was significantly greaterthan both AVLO (7.37±1.33 mg) and BASE (11.07±2.86 mg). Increasingconcentrations of 23RGD in the accelerant also increased the dry mass ofprecipitate with BASE and RHI both significantly higher than RVLO at(FIG. 18B). For example, BASE at 11.07±2.86 mg, RHI at 15.61±3.62 mg,and RMED at 10.2±1.42 mg were all significantly higher than RLO at1.9±0.6 mg. Increasing silk fibroin concentration increased precipitatedry mass with BASE being greater than SOLO and significantly greaterthan SCVLO (FIG. 18C). For example, BASE was greater than SOLO at7.84±1.49 mg and significantly greater than SCVLO at 4.15±1.0 mg.Increasing the percentage of ethanol in the accelerant solution alsoincreased dry mass of the precipitates with BASE producing significantlymore than ECLO (FIG. 18D). For example, BASE produced significantly morethan ECLO at 2.8±0.91 mg. ECVLO produced only trace quantities ofprecipitate (visible, but not recoverable in measurable quantities).These results indicate that greater concentrations of reactants (i.e.,accelerant solution, RGD, silk and ethanol) all increased the quantityof precipitant resultant.

The percent water in silk precipitates was determined as the percentageof mass lost after silk precipitates of each formulation types weresubjected to a lyophilization step. Increasing the volumetric fractionof accelerant added to make silk precipitates did not significantly(p≦0.05) affect the dry mass fraction of the resultant precipitates(FIG. 19A). For example, AVLO at (85.57±2.32%, BASE at 88.99±0.8%, andAVHI was 86.83±1.95%. Increasing concentrations of 23RGD in theaccelerant showed a significant increase in dry mass percentage withRVLO significantly less than RLO, RHI, and BASE (FIG. 19B). For example,RLO at 95.01±1.76% retained significantly more water than RMED at86.52±2.67%, RHI at 88.39±0.98%, and BASE. Increasing concentrations ofsilk fibroin did not result in a clear trend although SOLO wassignificantly greater than both SCVLO and BASE (FIG. 19C). For example,SOLO at 80.77±1.97% was significantly less than both SCVLO at86.94±1.98% and BASE. Increasing the percentage of ethanol in theaccelerant solution significantly decreased the dry mass percentage withECLO compared to BASE (FIG. 19D). For example, ECLO at 86.97±1.16%compared to BASE. In summary, greater concentrations of reactants (i.e.,accelerant solution, 23RGD, silk and ethanol) increased the quantity ofresultant precipitate. It is also of interest that there weresignificant differences between the dry mass fractions of BASE and bothRVLO and ECLO, possibly indicating different protein structures. Thesediffering protein structures might be more hydrophobic than BASE in thecase of ECLO and more hydrophilic in the case of RVLO. These propertiesmight used to affect the stability of the gels in an in vivo environmentwith more hydrophilic materials being more readily bioresorbed by thehost while more hydrophobic materials prove more resistant.

In examining the percent of water in the precipitates it is ofparticular interest that there were significant differences between BASEand both RLO and ECLO. This may result from structural motifs differentthan other precipitate types generated by RLO and ECLO. With respect toECLO, it has a greater proportion of β-sheet structure than BASE andwould be expected to entrain less water. However, the differenceobserved between RLO and base is difficult to explain. RLO has a greaterextent of β-sheet structure with less α-helix and random coil motifsthan BASE, yet it entrains a greater quantity of water. In fact, thissame trend is seen when comparing RLO to RMED, BASE, and RHI. Thesituation is further confounded in examining the relationship betweenthe initial secondary structures of RMED, BASE and RHI, as all initiallyexhibit greater quantities of α-helix and random coil than RLO, yet allentrain significantly less water. SOLO samples also had a significantlyhigher dry mass percentage as compared to BASE and SCVLO sample with noclear trend or reason for this occurrence. These data indicate thatthere may be a structural difference in these precipitates not apparentin the secondary structure of the materials which is affecting themanner in which the precipitates associate with water. It may be thecase that the RGD bound to these precipitates has altered in somefashion the manner in which the silk molecules are presented to water,enhancing their ability to associate with it.

Example 14 Gel Precipitate FTIR Spectrum Capture

Gel precipitates of each type, as described in Example 12, were analyzedby attenuated total reflectance Fourier-transform infrared (ATR-FTIR)spectroscopy using a Bruker Equinox 55 spectrophotometer (Bruker Optics,Inc., Billerica, Mass.) coupled with a Pike MIRACLE™ germanium crystal(PIKE Technologies, Madison, Wis.). Sample ATR signal spectra wereobtained by performing a 128-scan integration. Resolution was set to 4cm⁻¹ with a 1 cm⁻¹ interval from a range of 4000 to 400 cm⁻¹. FTIRspectra of pure water were also collected and subtracted manually fromthe gel spectra to remove confounding water signal at a ratio conduciveto flattening the region between 1800 cm⁻¹ and 1700 cm⁻¹ on thespectrum. After subtraction, the Amide I bands (1700-1600 cm⁻¹) ofrepresentative spectra were evaluated against characteristic peakscommonly accepted to be associated with secondary protein structures.

Examination of the silk precipitates under FTIR revealed that increasingthe volumetric ratio of accelerant added to the silk solution had littleeffect on their protein secondary structure (FIG. 20A). AVLO, BASE, andAVHI all exhibited similar characteristics with characteristic peaksaround 1624 cm⁻¹ and a toe region at 1698 cm⁻¹ indicating a predominanceof β-sheet and β-turn structure respectively. Each sample also exhibitedadditional structures at 1677 cm⁻¹, 1663 cm⁻¹, 1654 cm⁻¹ and 1645 cm⁻¹denoting additional interspersed β-sheet, β-turn, α-helical and randomcoil conformations respectively. Increasing concentrations of 23RGD inthe accelerant decreased β-sheet stability indicated by a peak shiftfrom ˜1621 cm⁻¹ in RVLO to ˜1624 cm⁻¹ in RLO (FIG. 20B). Furtherincreasing the concentration of 23RGD in BASE and RHI caused thisweakened β-sheet again accompanied by an increase in higher signalvalues in the 1654 cm⁻¹ and 1645 cm⁻¹ ranges, indicating increasedrandom coil and α-helical constituents. Otherwise, RVLO, RLO, BASE, andRHI revealed similar structures with dominant peaks in the 1620 cm⁻¹range and a toe region at 1698 cm⁻¹ with additional structures at 1654cm⁻¹ and 1645 cm⁻¹. Increasing concentrations of silk fibroin had littleperceptible effect on protein secondary structure (FIG. 20C). Thespectra for SCVLO, SOLO, and BASE each exhibited similar characteristicpeaks around 1624 cm⁻¹ with toe regions at 1698 cm¹ indicating apredominant β-sheet structure with additional α-helical and random coilconformations interspersed. Increasing the percentage of ethanol in theaccelerant solution resulted in less evidence of α-helical and randomcoil conformations indicated by a decrease in the signal between 1670cm⁻¹ and 1630 cm⁻¹ in both ECLO and BASE samples relative to ECVLO (FIG.20D). This decrease in α-helical and random coil is accompanied by anincrease in β-sheet structure.

Substantial similarity existed between all groups except for RVLO andECVLO, which each differ from BASE formulation. Each of these materialtypes exhibited a different secondary structure from both each other andfrom BASE, reinforcing the trend observed previously in the percent drymass of the precipitates. Higher concentrations of 23RGD yielded lessorganized β-sheet structures and lower concentrations of ethanol yieldedgreater quantities of α-helix and random coil motifs. It is possiblethat used in conjunction with one another, these two phenomena could beadjusted to develop silk structures resulting from silk solutions in anyof a variety of different protein conformations. These conformationscould, in turn, be tailored based upon the desired ultimate bulkproperties of the silk material.

It is expected that higher β-sheet components might provide the gel withgreater resistance to bioresorption and compressive loading, while atthe same time, making the material more rigid.

Example 15 Congo Red Staining of Gel Precipitates

Silk precipitate samples were stained with 100 μM Congo red in purifiedwater. Silk precipitate samples weighing 5-10 mg were vortexed with 500μL of this solution for 15 seconds, allowed to stand at room temperature(˜20-24° C.) for 10 minutes, then centrifuged at 16,000 g (RCF) for 10minutes. The supernatant was discarded and the pellet re-suspended byvortexing for 30 seconds in 1 mL of purified water. The process ofsoaking, centrifugation, aspirating and rinsing was repeated 3 times.The final pellet was removed, smeared on a glass microscope slide, andimaged under white and polarized light using a Microscope PC MACROFIRE™Model S99831 Camera (Optronics, Goleta, Calif.) and PICTUREFRAME™ 2.1software (Optronics, Goleta, Calif.) and a System Microscope Model BX41(Olympus, Melville, N.Y.).

None of the silk precipitate types exhibited the emerald luminescencetypically associated with amyloid fibrillar structures (FIG. 21). Allprecipitate types did exhibit bright white luminescence, indicative of arobust crystalline structure. The extent of this brightness does notappear to vary substantially by formulation, but only by sample quantityon the slide. Based on these results, it is unlikely that any of theseprecipitate types is amyloid in nature, a positive sign, as amyloidfibrils are associated with a number of negative pathologies in humans.

Example 16 23RGD Quantification in Gel Precipitates by HPLC

The amount of 23RGD bound to silk precipitates was quantified byanalyzing lyophilized samples. The 23RGD was removed by incubating thesamples for 4 hours in a dissolving buffer, then centrifuging on anEppendorf 5415C (Eppendorf North America Inc., Westbury, N.Y.) at 16,000g (RCF) for 30 minutes and the supernatant collected. This supernatantwas then centrifuged in identical fashion and the final supernatantcollected for HPLC analysis using a PerkinElmer Series 200 (PerkinElmer,Waltham, Mass.). The 23RGD peak areas from each curve were comparedagainst a standard curve. 1-Way ANOVA was used to test for significantdifferences caused by changing silk concentration, 23RGD concentration,and accelerant volume. A Student's t-test was used to test thesignificance of differences resulting from altered ethanolconcentrations.

Increasing the quantity of 23RGD/ethanol accelerant added resulted in asignificant increase (p 0.05) in 23RGD:silk ratio for both BASE and AVHIas compared to AVLO (FIG. 22A). For example, BASE at 8.7±0.6 and AVHI at10.5±1.2 were significantly increased as compared to AVLO at 5.2±1.8.Increasing the quantity of 23RGD in the accelerant solution resulted insignificant increases in 23RGD:silk ratio for each of RVLO, RLO, BASE,and RHI relative to each other (FIG. 22B). For example, RLO at 1.1±0.2,RMED at 6.95±0.49, BASE and RHI at 10.7±0.8 relative to each other.Changing the starting concentration of silk in solution prior toprecipitation did not affect 23RGD:silk ratio as those in SCVLO, SOLO,and BASE did not differ significantly (FIG. 22C). For example, SCVLO at11.0±0.4, SOLO at 9.9±1.8, and BASE did not differ significantly.Decreasing the ethanol content in the accelerant did not produce asignificant effect as observed by comparing ECLO and BASE (FIG. 22D).

Reviewing this data in light of the precipitate dry massing data, noneof the conditions explored resulted in isolation of silk (˜10-35%precipitated) nor 23RGD (˜5-30% precipitated) as limiting reagents inthe reaction. Precipitate samples generated at a calculated 10:123RGD:silk ratio consistently generated a “correct” molecular bindingratio. In the case of AVHI, this runs contrary to the trend of bound23RGD concentrations being approximately double the projected values asindicated by AVLO and BASE (about 5:1 and about 9:1, respectively). Thismight be explained by saturation of the silk with 23RGD in the case of10:1 23RGD precipitates. This is further reinforced by the behavior ofSCVLO and 0.6S 3R 10:1, both of which were produced using 2.45 mg/mL23RGD in 90% ethanol as the AVHI was. Both materials projected to havegreater than 10:1 ratios of bound 23RGD (20:1 and 13.4:1, respectively)based on the behavior of AVLO and BASE, but which both reached onlyabout 10:1 ratios. RHI, generated using a 4.5 mg/mL 23RGD concentrationin the accelerant which conceivably should have been high enough toinduce the postulated dimeric 23RGD reached only the expected 23RGDratio of about 10:1 not the postulated 20:1.

Few of the silk precipitates entrained a molar ratio similar to what wasinitially calculated (FIG. 23). Four groups, SCVLO, AVHI, RHI, and RLOcontained ratios similar to their calculated values of RGD per mole ofsilk. The six remaining groups contained ratios substantially greaterthan their calculated values. In the cases of AVLO, BASE, RMED, andSOLO, the RGD quantities were about 2-fold greater than expected.Although not wishing to be limited by theory, this greater observedmolar ratio may be indicative of the formation of a RGD bi-layer. It maybe the case that either micelles or lamellar structures of RGD existedin the 90% ethanol solution prior to addition to the silk, uponcontacting the aqueous phase, micellar stability was disrupted. As aresult, a bi-layer of RGD was formed at the solution interface, wherethese molecules began to interact with the silk molecules. The RLOsamples were made with a RGD concentration of 0.49 mg/mL in theaccelerant, the lowest used in this study and potentially within thesolubility range of RGD in 90% ethanol. RMED samples used 1.47 mg/mL andmost other formulations were made with a RGD accelerant concentration of2.45 mg/mL, above the RGD concentration at which dimerization becamefavorable in the solution. Further highlighting the possibility of RGDdimerizing in the ethanol solution is the behavior of ECLOprecipitation. The RGD concentration remains 2.45 mg/mL as with BASE andAVLO but the water concentration in the accelerant is increased to 20%and results in a binding of about 1.5-fold the expected total of RGD.This may be due to a decreased driving force for RGD bi-layer formationat the solution interface caused by the lower ethanol content. Thismight in turn cause disruption to fewer micellar structures in theinitial accelerant solution. It could also be explained by alteredmicellar structure, varying between a single peptide layer and amulti-lamellar structure depending upon the concentrations of water andethanol in the accelerant phase.

Precipitate samples generated at a calculated 10:1 RGD:silk ratioconsistently generated a “correct” molecular binding ratio. In the caseof AVHI, this runs contrary to the trend of bound RGD concentrationsbeing approximately double the projected values as indicated by AVLO andBASE (about 5:1 and about 9:1 respectively). It is possible that thismight be explained by saturation of the silk with RGD in the case of10:1 RGD precipitates. This is further reinforced by the behavior ofSCVLO and 0.6S 3R 10:1, both of which were produced using 2.45 mg/mL RGDin 90% ethanol as was AVHI. Both materials projected to have greaterthan 10:1 ratios of bound RGD (20:1 and 13.4:1 respectively) based onthe behavior of AVLO and BASE, but which both reached only about 10:1ratios. RHI, generated using a 4.5 mg/mL RGD concentration in theaccelerant which conceivably should have been high enough to induce thepostulated dimeric RGD, reached only the expected RGD ratio of about10:1 not the postulated 20:1. This may be attributed to the mode ofbinding between the silk molecules and the RGD molecules. It is expectedthat RGD will bind through a hydrophobic association mechanism anddespite the largely hydrophobic sequence of silk, it may be possiblethat there are approximately 5 sites which offer preferable RGD bindingstability. This presumption stems from the apparent saturation at 10:1RGD molecules per molecule of silk. Dependent upon the nature of RGDself-association at the solution boundary, it may be a case where singleRGD molecules or RGD dimers bind to these sites.

There are a series of properties further indicating the possibility of aspecific molecular assembly interaction between the silk and 23RGDaccelerant. Conspicuously, that 23RGD does localize to the precipitatesin a greater-than-calculated ratio but that it binds at intuitiveconcentrations which can be related quickly to the initially calculatedmolar ratios. The fact that this occurs without fully depleting eitherthe 23RGD or the silk fibroin molecules is of further interest. The FTIRdata also indicated that use of 0.49 mg/mL 23RGD in RVLO precipitatesinduced formation of distinctly different structures than use of 2.45mg/mL in BASE or 4.9 mg/mL in RHI which appeared similar to each other.RMED precipitates generated with 1.47 mg/mL of 23RGD containedcharacteristics of both RVLO and BASE/RHI material spectra. FTIRindicated a different structure from a 2.45 mg/mL of 23RGD in 70%ethanol accelerant in the case of ECVLO. These outcomes were bothreinforced in examining the percentage of dry mass from the resultantprecipitates (though ECLO is used to illustrate the trend in 23RGDsolubility in ethanol solution instead of ECVLO). Both of these assaysindicate the formation of different precipitate structures based uponthe extent of 23RGD saturation in the ethanol solution, conceivablyresulting from dimeric 23RGD binding or monomeric 23RGD binding.

This phenomenon likely results from the amphiphilic nature of 23RGD andthe varied chemistry of the solution phase between heavily ethanolic andheavily aqueous. It is possible that the hydrophilic ends of two 23RGDmolecules associate in the 90% ethanol solution, exposing the twohydrophobic ends to solution. Addition of this accelerant solution withdimeric 23RGD causes rapid association of the exposed hydrophobic endsof the 23RGD with hydrophobic domains of the silk molecules, rapidlyprecipitating them. This process occurs until the 90% ethanol accelerantsolution is sufficiently diluted with the aqueous silk solution to causethe dimeric assembly of the 23RGD molecules to no longer be favorable,as a result stopping precipitation. Based upon the apparent saturationat about 10 for 23RGD:silk ratio, there may also be a maximum of 5binding sites for the 23RGD dimer per molecule of silk. This knowledgemay be used to bind specific quantities of 23RGD to silk, while at thesame time dictating silk gel structure and resultant behavior.Additionally, this method may also potentially be applied to otheramphiphilic peptides of interest during their integration into a silkgel material.

These results indicate that silk precipitate quantity may be increasedby increasing the quantity of any reactant in the RGDEEG system. Silkprecipitates occurring during RGDEEG gelation are unlikely to beamyloid. Silk precipitate β-sheet structure may be increased by higherconcentrations of ethanol accelerant or lower concentrations of RGD. RGDmolecules may self-associate into micelles, lamellar structures, ordimers when placed into a strongly ethanolic solution, in turn,assembling with silk in a dimeric fashion during RGDEEG gelation. Silkmolecules may become saturated with RGD once they have bound about 10molecules. Silk precipitate structures may be altered by changing RGDconcentrations added, though the extent and nature of these changesremains unclear, as they are not perceptible in material secondarystructure. These altered structures may account for otherwiseunexplained increased appearance of α-helix and random coil motifs athigh RGD concentrations in precipitates. These altered structures mayaccount for otherwise unexplained increased resistance to proteolyticbioresorption of α-helix and random coil motifs at high RGDconcentrations in precipitates.

Example 17 Enzymatic Bioresorption of Gel Precipitates

A single sample of precipitate types selected for distinctly differentbehaviors from BASE in the previously listed assays including RVLO, RLO,BASE, RHI, ECLO, 0.6S 3R 5:1 weighing approximately 60 mg were massedusing an S-215 balance. These samples were placed in a solution ofProtease Type XIV from Streptomyces griseus (Sigma catalog no. P-5147)in phosphate buffered saline (PBS) was generated at a concentration of0.3 mg/mL (activity was 4.5 U/mg) at a ratio of 1 mL of proteasesolution per 100 mg of silk precipitate. The gel and protease solutionwere incubated for 24 hours at 37° C. with no mechanical mixing. After24 hours, the residual precipitate was isolated by straining throughstainless steel cloth as before and the specimens analyzed by FTIR asdescribed.

Accelerant quantity added did not substantially affect the bioresorptionbehavior of the materials as BASE, AVHI and AVLO all featured decreasedlevels of α-helix and random coil motifs (FIG. 23A). This decrease wasslightly larger in the case of AVLO which also featured a peak shiftfrom 1624 cm⁻¹ to 1622 cm⁻¹, indicating a more stable β-sheet structure.The 23RGD concentration did not appear to affect bioresorption behaviorof the materials either as RVLO, RLO, BASE and RHI all showed decreasedin α-helix and random coil motifs, though a greater portion of α-helixand random coil remained intact in RHI (FIG. 23B). However, a greaterportion of α-helix and random coil remained intact in RHI at Day 2relative to the other samples. Silk concentration did not substantiallyaffect the bioresorption behavior of the materials as BASE and SOLOexhibited decreased levels of α-helix and random coil motifs andfeatured slight peak shifts from 1624 cm⁻¹ to 1623 cm⁻¹ (FIG. 23C).

Despite differences in initial structures, all precipitate typesbioresorbed in a similar fashion with α-helix and random coil motifsdegraded preferentially to β-sheet. Only increasing the concentration of23RGD, as in the case of RHI, appeared to have any appreciable effect onthe final secondary structure of the precipitates. This appears to be acase where there is simply more α-helix and random coil structure uponinitial formation of these materials and they take more time to degradeto a similar extent of β-sheet structure as the other formulations. Useof this knowledge in conjunction with an ability to manipulate thesecondary protein structures of these materials could lead tobiomaterials with very specific lifetimes in vivo.

We claim:
 1. A formulation comprising silk conjugated to hyaluronicacid.
 2. The formulation of claim 1 wherein the silk is silk fibroin orthe silk is a silk molecule.
 3. The formulation of claim 2 wherein thehyaluronic acid is covalently attached to the silk.
 4. The formulationof claim 3 wherein the formulation further comprises an amino acidspacer which covalently attaches the hyaluronic acid to the silk.
 5. Theformulation of claim 4 wherein the amino acid spacer is selected fromthe group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22,SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25.